Anti-pd-1 antibodies, activatable anti-pd-1 antibodies, and methods of use thereof

ABSTRACT

The invention relates generally to antibodies that specifically bind programmed cell death protein 1 (PD-1), activatable antibodies that specifically bind to PD-1 and methods of making and using these anti-PD-1 antibodies and anti-PD-1 activatable antibodies in a variety of therapeutic, diagnostic and prophylactic indications.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/209,385, filed on Jul. 13, 2016, which claims the benefit of U.S. Provisional Application No. 62/191,902, filed Jul. 13, 2015; U.S. Provisional Application No. 62/205,825, filed Aug. 17, 2015; U.S. Provisional Application No. 62/295,314, filed Feb. 15, 2016; U.S. Provisional Application No. 62/323,543, filed Apr. 15, 2016 and U.S. Provisional Application No. 62/333,629, filed May 9, 2016; the contents of each of which are incorporated herein by reference in their entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: CYTM_044_001_US_SubSeqList_ST25.txt, dated recorded Mar. 21, 2019, file size 1.37 MB) in U.S. patent application Ser. No. 15/209,385.

FIELD OF THE INVENTION

The invention relates generally to antibodies that specifically bind programmed cell death protein 1 (PD-1), activatable antibodies that specifically bind to PD-1 and methods of making and using these anti-PD-1 antibodies and anti-PD-1 activatable antibodies in a variety of therapeutic, diagnostic and prophylactic indications.

BACKGROUND OF THE INVENTION

Antibody-based therapies have proven effective treatments for several diseases but in some cases, toxicities due to broad target expression have limited their therapeutic effectiveness. In addition, antibody-based therapeutics have exhibited other limitations such as rapid clearance from the circulation following administration.

Under conditions of chronic stimulation, T cells upregulate and sustain expression of the inhibitory receptor PD-1 to negatively regulate the quality and magnitude of T cell responses. The primary ligand for PD-1, PD-L1 is upregulated on many tumor cells and has been associated with inhibition of anti-tumor T-cell immunity via its engagement of PD-1 on tumor-infiltrating T cells. Clinical trials have confirmed the capacity of antibody blockade of either PD-1 or PD-L1 to restore the activity of durable tumor-specific immunity in patients across multiple tumor types. (Herbst et al, 2014; Lipson et al, 2015). However, because similar mechanisms control anti-tumor immunity and self-tolerance, systemic delivery of these checkpoint-targeted therapies can also induce systemic autoimmunity that can be exacerbated with combination treatments; such as nivolumab or pembrolizumab (anti-PD-1) and ipilimumab (anti-CTLA4). New approaches are therefore needed that provide anti-tumor activity without deregulating systemic immunity.

In the realm of small molecule therapeutics, strategies have been developed to provide prodrugs of an active chemical entity. Such prodrugs are administered in a relatively inactive (or significantly less active) form. Once administered, the prodrug is metabolized in vivo into the active compound. Such prodrug strategies can provide for increased selectivity of the drug for its intended target and for a reduction of adverse effects.

Accordingly, there is a continued need in the field of antibody-based therapeutics for antibodies that mimic the desirable characteristics of the small molecule prodrug.

SUMMARY OF THE INVENTION

The disclosure provides antibodies or antigen-binding fragments thereof that specifically bind programmed cell death protein 1 (PD-1), also known as CD279, SLEB2, and/or hSLE1. The use of the term “PD-1” is intended to cover any variation thereof, such as, by way of non-limiting example, PD1 and/or PD 1, all variations are used herein interchangeably. Aberrant expression and/or activity of PD-1 and PD-1-related signaling has been implicated in the pathogenesis of many diseases and disorders, such as cancer.

The present invention provides monoclonal antibodies (mAbs), activatable antibodies, and antigen-binding fragments thereof that specifically bind PD-1.

In some embodiments, the antibody includes an antibody or antigen-binding fragment thereof that specifically binds PD-1. In some embodiments, the antibody or antigen-binding fragment thereof that binds PD-1 is a monoclonal antibody, domain antibody, single chain, Fab fragment, a F(ab′)₂ fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody. In some embodiments, such an antibody or antigen-binding fragment thereof that binds PD-1 is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody.

In some embodiments, the antibody includes an isolated antibody or antigen binding fragment thereof (AB) that specifically binds to mammalian PD-1, wherein the AB has one or more of the characteristics selected from the group consisting of: (a) the AB inhibits binding of mammalian PD-1 to mammalian PDL1 with an EC₅₀ value less than 5 nM; (b) the AB inhibits binding of mammalian PD-1 to mammalian PDL2 with an EC₅₀ value less than 5 nM; and (c) the AB specifically binds to human PD-1 and cynomolgus monkey PD-1.

In some embodiments, the antibody specifically binds to the mammalian PD-1 with a dissociation constant of 0.01 nM to 5 nM, 0.05 nM to 5 nM, 0.1 nM to 5 nM, 0.2 nM to 5 nM, 0.3 nM to 5 nM, 0.4 nM to 5 nM, 0.5 nM to 5 nM, 0.75 nM to 5 nM, 1 nM to 5 nM, 2 nM to 5 nM, 0.01 nM to 2 nM, 0.05 nM to 2 nM, 0.1 nM to 2 nM, 0.2 nM to 2 nM, 0.3 nM to 2 nM, 0.4 nM to 2 nM, 0.5 nM to 2 nM, 0.75 nM to 1 nM, 1 nM to 2 nM, 0.01 nM to 1 nM, 0.05 nM to 1 nM, 0.1 nM to 1 nM, 0.2 nM to 1 nM, 0.3 nM to 1 nM, 0.4 nM to 1 nM, 0.5 nM to 1 nM, 0.75 nM to 1 nM, 0.01 nM to 0.75 nM, 0.05 nM to 0.75 nM, 0.1 nM to 0.75 nM, 0.2 nM to 0.75 nM, 0.3 nM to 0.75 nM, 0.4 nM to 0.75 nM, 0.5 nM to 0.75 nM, 0.01 nM to 0.5 nM, 0.05 nM to 0.5 nM, 0.1 nM to 0.5 nM, 0.2 nM to 0.5 nM, 0.3 nM to 0.5 nM, 0.4 nM to 0.5 nM, 0.01 nM to 0.4 nM, 0.05 nM to 0.4 nM, 0.1 nM to 0.4 nM, 0.2 nM to 0.4 nM, 0.3 nM to 0.4 nM, 0.01 nM to 0.3 nM, 0.05 nM to 0.3 nM, 0.1 nM to 0.3 nM, 0.2 nM to 0.3 nM, 0.01 nM to 0.2 nM, 0.05 nM to 0.2 nM, 0.1 nM to 0.2 nM, 0.01 nM to 0.1 nM, 0.05 nM to 0.1 nM, or 0.01 nM to 0.05 nM.

In some embodiments, the mammalian PD-1 is selected from the group consisting of a human PD-1 and a cynomolgus monkey PD-1. In some embodiments, the mammalian PD-1 is a murine PD-1. In some embodiments, the antibody specifically binds to human PD-1 or cynomolgus monkey PD-1 with a dissociation constant of less than or equal to 1 nM. In some embodiments, the mammalian PD-1 is a human PD-1.

In some embodiments, the antibody or antigen binding fragment thereof specifically binds to the mammalian PD-1 with a dissociation constant is less than or equal to 0.01 nM, less than or equal to 0.05 nM, less than or equal to 0.1 nM, less than or equal to 0.2 nM, less than or equal to 0.3 nM, less than or equal to 0.4 nM, less than or equal to 0.5 nM, less than or equal to 0.75 nM, and less than or equal to 1 nM.

In some embodiments, the antibody has one or more of the characteristics selected from the group consisting of: (a) the AB specifically binds human PD-1 and cynomolgus monkey PD-1; (b) the AB inhibits binding of human PDL1 and human PDL2 to human PD-1; (c) the AB inhibits binding of cynomolgus monkey PDL1 and cynomolgus monkey PDL2 to cynomolgus monkey PD-1; (d) the AB specifically binds to murine PD-1; and (e) the AB inhibits binding of murine PDL1 and murine PDL2 to murine PD-1.

In some embodiments, the antibody blocks the ability of a natural ligand to bind to the mammalian PDL1 with an EC₅₀ of 0.1 nM to 10 nM, 0.1 nM to 5 nM, 0.1 nM to 3 nM, 0.1 nM to 2 nM, 0.1 nM to 1 nM, 0.1 nM to 0.5 nM, 0.1 nM to 0.25 nM, 0.25 nM to 10 nM, 0.25 nM to 5 nM, 0.25 nM to 3 nM, 0.25 nM to 2 nM, 0.25 nM to 1 nM, 0.25 nM to 0.5 nM, 0.5 nM to 10 nM, 0.5 nM to 5 nM, 0.5 nM to 3 nM, 0.5 nM to 2 nM, 0.5 nM to 1 nM, 1 nM to 10 nM, 1 nM to 5 nM, 1 nM to 3 nM, 1 nM to 2 nM, 2 nM to 10 nM, 2 nM to 5 nM, 2 nM to 3 nM, 3 nM to 10 nM, 3 nM to 5 nM, or 5 nM to 10 nM. In some embodiments, the natural ligand is a mammalian PDL1 or a mammalian PDL2. In some embodiments, the natural ligand is selected from the group consisting of: a human PDL1, a human PDL2, a cynomolgus monkey PDL1, and a cynomolgus monkey PDL2. In some embodiments, the natural ligand is a murine PDL1 or a murine PDL2.

In some embodiments, the antibody blocks the ability of a natural ligand to bind to the mammalian PDL1 with an EC₅₀ of less than or equal to 0.1 nM, less than or equal to 0.25 nM, less than or equal to 0.5 nM, less than or equal to 1 nM, less than or equal to 2 nM, less than or equal to 3 nM, less than or equal to 4 nM, less than or equal to 5 nM or less than or equal to 10 nM.

In some embodiments, the anti-PD-1 antibody includes a heavy chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the anti-PD-1 antibody includes a heavy chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the anti-PD-1 antibody includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21. In some embodiments, the anti-PD-1 antibody includes a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 45 or SEQ ID NO: 47. In some embodiments, the anti-PD-1 antibody includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21 and a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 45 or SEQ ID NO: 47. In some embodiments, the anti-PD-1 antibody includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21 and a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 45. In some embodiments, the anti-PD-1 antibody includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21 and a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 47.

In some embodiments, the anti-PD-1 antibody includes the combination of the complementarity determining region (CDR) sequences of the heavy chain amino acid sequence of SEQ ID NO: 21. In some embodiments, the anti-PD-1 antibody includes the combination of the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 45 or of SEQ ID NO: 47. In some embodiments, the anti-PD-1 antibody includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 21, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 45 or 47. In some embodiments, the anti-PD-1 antibody includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 21, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 45. In some embodiments, the anti-PD-1 antibody includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 21, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 47.

In some embodiments, the anti-PD-1 antibody includes: (a) a variable heavy chain complementarity determining region 1 (VH CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 653-657; (b) a variable heavy chain complementarity determining region 2 (VH CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 658-663; (c) a variable heavy chain complementarity determining region 3 (VH CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 664-668; (d) a variable light chain complementarity determining region 1 (VL CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 669-677; (e) a variable light chain complementarity determining region 2 (VL CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 678-682; and (f) variable light chain complementarity determining region 3 (VL CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 683-687.

In some embodiments, the anti-PD-1 antibody includes a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664).

In some embodiments, the anti-PD-1 antibody includes a combination of a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675) or RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, the anti-PD-1 antibody includes a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, the anti-PD-1 antibody includes a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VL CDR1 sequence comprises RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683).

In some embodiments, the anti-PD-1 antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664); the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675) or RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, the anti-PD-1 antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664); the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, the anti-PD-1 antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664); the VL CDR1 sequence comprises RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683).

In some embodiments, the anti-PD-1 antibody includes a heavy chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 1346, and a light chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 626.

In some embodiments, the anti-PD-1 antibody includes a heavy chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 1514, and a light chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 638.

In some embodiments, the anti-PD-1 antibody includes the combination of the complementarity determining region (CDR) sequences of the heavy chain amino acid sequence of SEQ ID NO: 1346. In some embodiments, the anti-PD-1 antibody includes the combination of the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 626. In some embodiments, the anti-PD-1 antibody includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 1346, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 626.

In some embodiments, the anti-PD-1 antibody includes a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, wherein the VH CDR1 sequence comprises GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises IWYDGSKR (SEQ ID NO: 1706); and the VH CDR3 sequence comprises TNDDY (SEQ ID NO: 1707).

In some embodiments, the anti-PD-1 antibody includes a combination of a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein the VL CDR1 sequence comprises QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises QQSSNWPRT (SEQ ID NO: 1710).

In some embodiments, the anti-PD-1 antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises IWYDGSKR (SEQ ID NO: 1706); the VH CDR3 sequence comprises TNDDY (SEQ ID NO: 1707); the VL CDR1 sequence comprises QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises QQSSNWPRT (SEQ ID NO: 1710).

In some embodiments, the anti-PD-1 antibody includes the combination of the complementarity determining region (CDR) sequences of the heavy chain amino acid sequence of SEQ ID NO: 1514. In some embodiments, the anti-PD-1 antibody includes the combination of the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 638. In some embodiments, the anti-PD-1 antibody includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 1514, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 638.

In some embodiments, the anti-PD-1 antibody includes a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, wherein the VH CDR1 sequence comprises GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises INPSNGGT (SEQ ID NO: 1712); and the VH CDR3 sequence comprises RRDYRFDMGFDY (SEQ ID NO: 1713).

In some embodiments, the anti-PD-1 antibody includes a combination of a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein the VL CDR1 sequence comprises KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises QHSRDLPLT (SEQ ID NO: 1716).

In some embodiments, the anti-PD-1 antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises INPSNGGT (SEQ ID NO: 1712); the VH CDR3 sequence comprises RRDYRFDMGFDY (SEQ ID NO: 1713); the VL CDR1 sequence comprises KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises QHSRDLPLT (SEQ ID NO: 1716).

In some embodiments, the antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37. In some embodiments, the antibody comprises a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, the antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37. In some embodiments, the antibody comprises a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, the antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, the anti-PD-1 antibody includes a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21, and a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 47. In some embodiments, the anti-PD-1 antibody includes a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21, and a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the anti-PD-1 antibody includes a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21, and a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 47.

In some embodiments, the anti-PD-1 antibody includes a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1346, and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 626.

In some embodiments, the anti-PD-1 antibody includes a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1514, and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 638.

In some embodiments, the antibody comprises a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence shown in Table 8; a VH CDR2 sequence shown in Table 8; a VH CDR3 sequence shown in Table 8; a VL CDR1 sequence shown in Table 8; a VL CDR2 sequence shown in Table 8; and a VL CDR3 sequence shown in Table 8.

In some embodiments, the antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence shown in Table 8; a VH CD2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence shown in Table 8; a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR3 sequence shown in Table 8; a VL CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR1 sequence shown in Table 8; a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence shown in Table 8; and a VL CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR3 sequence shown in Table 8.

In some embodiments, the antibody comprises a heavy chain that comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the combination is a combination of the three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single row in Table 8.

In some embodiments, the antibody comprises a light chain that comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination is a combination of the three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown in a single row in Table 8.

In some embodiments, the antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence shown in a single row in Table 8, and a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence shown in a single row in Table 8.

In some embodiments, the antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence shown in a single row in Table 8, and a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence shown in a single row in Table 8, wherein each CDR sequence in the combination comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the corresponding VH CDR sequence shown in a single row in Table 8 and the corresponding VL CDR shown in a single row in Table 8.

In some embodiments, the antibody comprises a heavy chain that comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein each CDR sequence in the combination comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the corresponding CDR sequence in a combination of three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single row in Table 8.

In some embodiments, the antibody comprises a light chain that comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein each CDR sequence in the combination comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the corresponding CDR sequence in a combination of three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown in a single row in Table 8.

In some embodiments, the antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to IWYDGSKR (SEQ ID NO: 1706); and the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TNDDY (SEQ ID NO: 1707).

In some embodiments, the anti-PD-1 antibody includes a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QQSSNWPRT (SEQ ID NO: 1710).

In some embodiments, the anti-PD-1 antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to IWYDGSKR (SEQ ID NO: 1706); the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TNDDY (SEQ ID NO: 1707); the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QQSSNWPRT (SEQ ID NO: 1710).

In some embodiments, the antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to INPSNGGT (SEQ ID NO: 1712); and the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to RRDYRFDMGFDY (SEQ ID NO: 1713).

In some embodiments, the anti-PD-1 antibody includes a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QHSRDLPLT (SEQ ID NO: 1716).

In some embodiments, the anti-PD-1 antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to INPSNGGT (SEQ ID NO: 1712); the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to RRDYRFDMGFDY (SEQ ID NO: 1713); the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QHSRDLPLT (SEQ ID NO: 1716).

In some embodiments, the antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37. In some embodiments, the antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37.

In some embodiments, the antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, the antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the anti-PD-1 is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 1346, and a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 626.

In some embodiments, the anti-PD-1 antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 1514, and a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 638.

In some embodiments, the antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37. In some embodiments, the antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37.

In some embodiments, the antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, the antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a nucleic acid sequence encoding a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the anti-PD-1 is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1346, and a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain t that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 626.

In some embodiments, the anti-PD-1 antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1514, and a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 638.

In some embodiments, the antibody is encoded by a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 18, 22, 24, 26, 28, 30, 32, 34, 36, and 38. In some embodiments, the antibody is encoded by a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 36, and 38.

In some embodiments, the antibody is encoded by a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, 8, 12, 16, 20, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60. In some embodiments, the antibody is encoded by a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60.

In some embodiments, the antibody is encoded by heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: selected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 18, 22, 24, 26, 28, 30, 32, 34, 36, and 38, and a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, 8, 12, 16, 20, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60. In some embodiments, the antibody is encoded by heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: selected from the group consisting of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 36, and 38, and a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60.

In some embodiments, the antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 18, 22, 24, 26, 28, 30, 32, 34, 36, and 38. In some embodiments, the antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 36, and 38.

In some embodiments, the antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, 8, 12, 16, 20, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60. In some embodiments, the antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60.

In some embodiments, the antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 18, 22, 24, 26, 28, 30, 32, 34, 36, and 38, and a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, 8, 12, 16, 20, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60. In some embodiments, the antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 36, and 38, and a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60.

In some embodiments, the antibody or antigen-binding fragment thereof is incorporated in a multispecific antibody or antigen-binding fragment thereof, where at least one arm of the multispecific antibody specifically binds PD-1. In some embodiments, the antibody or antigen-binding fragment thereof is incorporated in a bispecific antibody or antigen-binding fragment thereof, where at least one arm of the bispecific antibody specifically binds PD-1.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 45 or SEQ ID NO: 47. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21 and a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 45 or SEQ ID NO: 47. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21 and a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 45. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21 and a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 47.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes the combination of the complementarity determining region (CDR) sequences of the heavy chain amino acid sequence of SEQ ID NO: 21. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes the combination of the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 45 or of SEQ ID NO: 47. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 21, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 45 or 47. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 21, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 45. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 21, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 47.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody includes: (a) a variable heavy chain complementarity determining region 1 (VH CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 653-657; (b) a variable heavy chain complementarity determining region 2 (VH CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 658-663; (c) a variable heavy chain complementarity determining region 3 (VH CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 664-668; (d) a variable light chain complementarity determining region 1 (VL CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 669-677; (e) a variable light chain complementarity determining region 2 (VL CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 678-682; and (f) variable light chain complementarity determining region 3 (VL CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 683-687

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675) or RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VL CDR1 sequence comprises RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664); the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675) or RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664); the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664); the VL CDR1 sequence comprises RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a heavy chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 1346, and a light chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 626.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a heavy chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 1514, and a light chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 638.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes the combination of the complementarity determining region (CDR) sequences of the heavy chain amino acid sequence of SEQ ID NO: 1346. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes the combination of the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 626. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 1346, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 626.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, wherein the VH CDR1 sequence comprises GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises IWYDGSKR (SEQ ID NO: 1706); and the VH CDR3 sequence comprises TNDDY (SEQ ID NO: 1707).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein the VL CDR1 sequence comprises QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises QQSSNWPRT (SEQ ID NO: 1710).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises IWYDGSKR (SEQ ID NO: 1706); the VH CDR3 sequence comprises TNDDY (SEQ ID NO: 1707); the VL CDR1 sequence comprises QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises QQSSNWPRT (SEQ ID NO: 1710).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes the combination of the complementarity determining region (CDR) sequences of the heavy chain amino acid sequence of SEQ ID NO: 1514. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes the combination of the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 638. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 1514, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 638.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, wherein the VH CDR1 sequence comprises GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises INPSNGGT (SEQ ID NO: 1712); and the VH CDR3 sequence comprises RRDYRFDMGFDY (SEQ ID NO: 1713).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein the VL CDR1 sequence comprises KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises QHSRDLPLT (SEQ ID NO: 1716).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises INPSNGGT (SEQ ID NO: 1712); the VH CDR3 sequence comprises RRDYRFDMGFDY (SEQ ID NO: 1713); the VL CDR1 sequence comprises KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises QHSRDLPLT (SEQ ID NO: 1716).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1346, and a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 626.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1514, and a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 638.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to IWYDGSKR (SEQ ID NO: 1706); and the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TNDDY (SEQ ID NO: 1707).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QQSSNWPRT (SEQ ID NO: 1710).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to IWYDGSKR (SEQ ID NO: 1706); the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TNDDY (SEQ ID NO: 1707); the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QQSSNWPRT (SEQ ID NO: 1710).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to INPSNGGT (SEQ ID NO: 1712); and the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to RRDYRFDMGFDY (SEQ ID NO: 1713).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QHSRDLPLT (SEQ ID NO: 1716).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to INPSNGGT (SEQ ID NO: 1712); the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to RRDYRFDMGFDY (SEQ ID NO: 1713); the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QHSRDLPLT (SEQ ID NO: 1716).

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence shown in Table 8; a VH CDR2 sequence shown in Table 8; a VH CDR3 sequence shown in Table 8; a VL CDR1 sequence shown in Table 8; a VL CDR2 sequence shown in Table 8; and a VL CDR3 sequence shown in Table 8.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence shown in Table 8; a VH CD2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence shown in Table 8; a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR3 sequence shown in Table 8; a VL CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR1 sequence shown in Table 8; a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence shown in Table 8; and a VL CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR3 sequence shown in Table 8.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination is a combination of the six CDR sequences (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) shown in a single row in Table 8.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a heavy chain that comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the combination is a combination of the three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single row in Table 8.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence shown in a single row in Table 8, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence shown in a single row in Table 8, wherein each CDR sequence in the combination comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the corresponding VH CDR sequence shown in a single row in Table 8 and the corresponding VL CDR shown in a single row in Table 8.

In some embodiments, at least one arm of the multispecific antibody, e.g., a bispecific antibody, comprises a heavy chain that comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein each CDR sequence in the combination comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the corresponding CDR sequence in a combination of three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single row in Table 8.

The disclosure also provides activatable antibodies that include an antibody or antigen-binding fragment thereof that specifically binds PD-1 coupled to a masking moiety (MM), such that coupling of the MM reduces the ability of the antibody or antigen-binding fragment thereof to bind PD-1. In some embodiments, the MM is coupled via a cleavable moiety (CM) that includes sequence that functions as a substrate for a protease. The activatable anti-PD-1 antibodies of the disclosure are activated when the cleavable moiety is cleaved by a protease. For example, the protease is produced by a tumor that is in proximity to T cells that express PD-1. In some embodiments, the protease is produced by a tumor that is co-localized with T cells that express PD-1.

The activatable anti-PD-1 antibodies provided herein, also referred to herein as anti-PD-1 activatable antibodies or PD-1 activatable antibodies, are stable in circulation, activated at intended sites of therapy and/or diagnosis but not in normal, e.g., healthy tissue or other tissue not targeted for treatment and/or diagnosis, and, when activated, exhibit binding to PD-1 that is at least comparable to the corresponding, unmodified antibody.

The invention also provides methods of treating, preventing and/or delaying the onset or progression of, or alleviating a symptom associated with aberrant expression and/or activity of PD-1 in a subject using antibodies or activatable antibodies that bind PD-1, particularly activatable antibodies that bind and neutralize or otherwise inhibit at least one biological activity of PD-1.

In some embodiments, the activatable anti-PD-1 antibody comprises an activatable antibody that, in an activated state, specifically binds to mammalian PD-1, wherein said activatable antibody comprises: an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian PD-1; a masking moiety (MM) that inhibits the binding of the AB to mammalian PD-1 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.

In some embodiments, the activatable anti-PD-1 antibody comprises an activatable antibody that, in an activated state, (a) specifically binds to mammalian PD-1; and (b) specifically blocks a natural ligand of PD-1 from binding to the mammalian PD-1, wherein the activatable antibody comprises: an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian PD-1; a masking moiety (MM) that inhibits the binding of the AB to mammalian PD-1 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.

In some embodiments, the activatable antibody in an uncleaved state specifically binds to the mammalian PD-1 with a dissociation constant of 0.5 nM to 1 nM, 0.5 nM to 2 nM, 0.5 nM to 5 nM, 0.5 nM to 10 nM, 0.5 nM to 15 nM, 0.5 nM to 20 nM, 0.5 nM to 25 nM, 0.5 nM to 50 nM, 0.5 nM to 75 nM, 0.5 nM to 100 nM, 0.5 nM to 150 nM, 0.5 nM to 200 nM, 0.5 nM to 300 nM, 0.5 nM to 400 nM, 1 nM to 2 nM, 1 nM to 5 nM, 1 nM to 10 nM, 1 nM to 15 nM, 1 nM to 20 nM, 1 nM to 25 nM, 1 nM to 50 nM, 1 nM to 75 nM, 1 nM to 100 nM, 1 nM to 150 nM, 1 nM to 200 nM, 1 nM to 300 nM, 1 nM to 400 nM, 2 nM to 5 nM, 2 nM to 10 nM, 2 nM to 15 nM, 2 nM to 20 nM, 2 nM to 25 nM, 2 nM to 50 nM, 2 nM to 75 nM, 2 nM to 100 nM, 2 nM to 150 nM, 2 nM to 200 nM, 2 nM to 300 nM, 2 nM to 400 nM, 5 nM to 10 nM, 5 nM to 15 nM, 5 nM to 20 nM, 5 nM to 25 nM, 5 nM to 50 nM, 5 nM to 75 nM, 5 nM to 100 nM, 5 nM to 150 nM, 5 nM to 200 nM, 5 nM to 300 nM, 5 nM to 400 nM, 10 nM to 15 nM, 10 nM to 20 nM, 10 nM to 25 nM, 10 nM to 50 nM, 10 nM to 75 nM, 10 nM to 100 nM, 10 nM to 150 nM, 10 nM to 200 nM, 10 nM to 300 nM, 10 nM to 400 nM, 15 nM to 20 nM, 15 nM to 25 nM, 15 nM to 50 nM, 15 nM to 75 nM, 15 nM to 100 nM, 15 nM to 150 nM, 15 nM to 200 nM, 15 nM to 300 nM, 15 nM to 400 nM, 20 nM to 25 nM, 20 nM to 50 nM, 20 nM to 75 nM, 20 nM to 100 nM, 20 nM to 150 nM, 20 nM to 200 nM, 20 nM to 300 nM, 20 nM to 400 nM, 25 nM to 50 nM, 25 nM to 75 nM, 25 nM to 100 nM, 25 nM to 150 nM, 25 nM to 200 nM, 25 nM to 300 nM, 25 nM to 400 nM, 50 nM to 75 nM, 50 nM to 100 nM, 50 nM to 150 nM, 50 nM to 200 nM, 50 nM to 300 nM, 50 nM to 400 nM, 75 nM to 100 nM, 75 nM to 150 nM, 75 nM to 200 nM, 75 nM to 300 nM, 75 nM to 400 nM, 100 nM to 150 nM, 100 nM to 200 nM, 100 nM to 300 nM, 100 nM to 400 nM, 150 nM to 200 nM, 150 nM to 300 nM, 150 nM to 400 nM, 200 nM to 300 nM, 200 nM to 400 nM, or 300 nM to 400 nM.

In some embodiments, the activatable antibody in an activated state specifically binds to the mammalian PD-1 with a dissociation constant of 0.01 nM to 5 nM, 0.05 nM to 5 nM, 0.1 nM to 5 nM, 0.2 nM to 5 nM, 0.3 nM to 5 nM, 0.4 nM to 5 nM, 0.5 nM to 5 nM, 0.75 nM to 5 nM, 1 nM to 5 nM, 2 nM to 5 nM, 0.01 nM to 2 nM, 0.05 nM to 2 nM, 0.1 nM to 2 nM, 0.2 nM to 2 nM, 0.3 nM to 2 nM, 0.4 nM to 2 nM, 0.5 nM to 2 nM, 0.75 nM to 1 nM, 1 nM to 2 nM, 0.01 nM to 1 nM, 0.05 nM to 1 nM, 0.1 nM to 1 nM, 0.2 nM to 1 nM, 0.3 nM to 1 nM, 0.4 nM to 1 nM, 0.5 nM to 1 nM, 0.75 nM to 1 nM, 0.01 nM to 0.75 nM, 0.05 nM to 0.75 nM, 0.1 nM to 0.75 nM, 0.2 nM to 0.75 nM, 0.3 nM to 0.75 nM, 0.4 nM to 0.75 nM, 0.5 nM to 0.75 nM, 0.01 nM to 0.5 nM, 0.05 nM to 0.5 nM, 0.1 nM to 0.5 nM, 0.2 nM to 0.5 nM, 0.3 nM to 0.5 nM, 0.4 nM to 0.5 nM, 0.01 nM to 0.4 nM, 0.05 nM to 0.4 nM, 0.1 nM to 0.4 nM, 0.2 nM to 0.4 nM, 0.3 nM to 0.4 nM, 0.01 nM to 0.3 nM, 0.05 nM to 0.3 nM, 0.1 nM to 0.3 nM, 0.2 nM to 0.3 nM, 0.01 nM to 0.2 nM, 0.05 nM to 0.2 nM, 0.1 nM to 0.2 nM, 0.01 nM to 0.1 nM, 0.05 nM to 0.1 nM, or 0.01 nM to 0.05 nM.

In some embodiments, the activatable antibody comprises an AB that specifically binds to the mammalian PD-1 with a dissociation constant of 0.01 nM to 5 nM, 0.05 nM to 5 nM, 0.1 nM to 5 nM, 0.2 nM to 5 nM, 0.3 nM to 5 nM, 0.4 nM to 5 nM, 0.5 nM to 5 nM, 0.75 nM to 5 nM, 1 nM to 5 nM, 2 nM to 5 nM, 0.01 nM to 2 nM, 0.05 nM to 2 nM, 0.1 nM to 2 nM, 0.2 nM to 2 nM, 0.3 nM to 2 nM, 0.4 nM to 2 nM, 0.5 nM to 2 nM, 0.75 nM to 1 nM, 1 nM to 2 nM, 0.01 nM to 1 nM, 0.05 nM to 1 nM, 0.1 nM to 1 nM, 0.2 nM to 1 nM, 0.3 nM to 1 nM, 0.4 nM to 1 nM, 0.5 nM to 1 nM, 0.75 nM to 1 nM, 0.01 nM to 0.75 nM, 0.05 nM to 0.75 nM, 0.1 nM to 0.75 nM, 0.2 nM to 0.75 nM, 0.3 nM to 0.75 nM, 0.4 nM to 0.75 nM, 0.5 nM to 0.75 nM, 0.01 nM to 0.5 nM, 0.05 nM to 0.5 nM, 0.1 nM to 0.5 nM, 0.2 nM to 0.5 nM, 0.3 nM to 0.5 nM, 0.4 nM to 0.5 nM, 0.01 nM to 0.4 nM, 0.05 nM to 0.4 nM, 0.1 nM to 0.4 nM, 0.2 nM to 0.4 nM, 0.3 nM to 0.4 nM, 0.01 nM to 0.3 nM, 0.05 nM to 0.3 nM, 0.1 nM to 0.3 nM, 0.2 nM to 0.3 nM, 0.01 nM to 0.2 nM, 0.05 nM to 0.2 nM, 0.1 nM to 0.2 nM, 0.01 nM to 0.1 nM, 0.05 nM to 0.1 nM, or 0.01 nM to 0.05 nM.

In some embodiments, the mammalian PD-1 is selected from the group consisting of a human PD-1 and a cynomolgus monkey PD-1. In some embodiments, the AB specifically binds to human PD-1 or cynomolgus monkey PD-1 with a dissociation constant of less than or equal to 1 nM. In some embodiments, the mammalian PD-1 is a human PD-1. In some embodiments, the AB has one or more of the characteristics selected from the group consisting of: (a) the AB specifically binds human PD-1 and cynomolgus monkey PD-1; (b) the AB inhibits binding of human PDL1 and human PDL2 to human PD-1; and (c) the AB inhibits binding of cynomolgus monkey PDL1 and cynomolgus monkey PDL2 to cynomolgus monkey PD-1.

In some embodiments, the mammalian PD-1 is mouse PD-1. In some embodiments, the activatable antibody comprises an AB that specifically binds mouse PD-1 or inhibits binding of mouse PDL1 and mouse PDL2 to mouse PD1.

In some embodiments, the activatable antibody in an uncleaved state specifically binds to the mammalian PD-1 with a dissociation constant greater than or equal to 0.5 nM, greater than or equal to 1 nM, greater than or equal to 2 nM, greater than or equal to 3 nM, greater than or equal to 4 nM, greater than or equal to 5 nM, greater than or equal to 10 nM, greater than or equal to 15 nM, greater than or equal to 20 nM, greater than or equal to 25 nM, greater than or equal to 50 nM, greater than or equal to 75 nM, greater than or equal to 100 nM, greater than or equal to 150 nM, greater than or equal to 200 nM, greater than or equal to 300 nM and/or greater than or equal to 400 nM.

In some embodiments, the activatable antibody in an activated state specifically binds to the mammalian PD-1 with a dissociation constant less than or equal to 0.01 nM, less than or equal to 0.05 nM, less than or equal to 0.1 nM, less than or equal to 0.2 nM, less than or equal to 0.3 nM, less than or equal to 0.4 nM, less than or equal to 0.5 nM, less than or equal to 0.75 nM, and less than or equal to 1 nM.

In some embodiments, the activatable antibody comprises an AB that specifically binds to the mammalian PD-1 with a dissociation constant less than or equal to 0.01 nM, less than or equal to 0.05 nM, less than or equal to 0.1 nM, less than or equal to 0.2 nM, less than or equal to 0.3 nM, less than or equal to 0.4 nM, less than or equal to 0.5 nM, less than or equal to 0.75 nM, and less than or equal to 1 nM.

In some embodiments, the activatable antibody comprises an AB blocks the ability of a natural ligand to bind to the mammalian PDL1 with an EC₅₀ of 0.1 nM to 10 nM, 0.1 nM to 5 nM, 0.1 nM to 3 nM, 0.1 nM to 2 nM, 0.1 nM to 1 nM, 0.1 nM to 0.5 nM, 0.1 nM to 0.25 nM, 0.25 nM to 10 nM, 0.25 nM to 5 nM, 0.25 nM to 3 nM, 0.25 nM to 2 nM, 0.25 nM to 1 nM, 0.25 nM to 0.5 nM, 0.5 nM to 10 nM, 0.5 nM to 5 nM, 0.5 nM to 3 nM, 0.5 nM to 2 nM, 0.5 nM to 1 nM, 1 nM to 10 nM, 1 nM to 5 nM, 1 nM to 3 nM, 1 nM to 2 nM, 2 nM to 10 nM, 2 nM to 5 nM, 2 nM to 3 nM, 3 nM to 10 nM, 3 nM to 5 nM, or 5 nM to 10 nM.

In some embodiments, the natural ligand is a mammalian PDL1 or a mammalian PDL2. In some embodiments, the natural ligand is selected from the group consisting of: a human PDL1, a human PDL2, a cynomolgus monkey PDL1, and a cynomolgus monkey PDL2.

In some embodiments, the activatable antibody has one or more of the following characteristics: (a) the AB induces type 1 diabetes in a non-obese diabetic (NOD) mouse; and (b) the activatable antibody in an uncleaved state inhibits the induction of type 1 diabetes in a NOD mouse.

In some embodiments, the activatable antibody inhibits the induction of type 1 diabetes in the NOD mouse after administration of the activatable antibody at a single dose of 0.5 mg/kg to 15 mg/kg, 1 mg/kg to 15 mg/kg, 2 mg/kg to 15 mg/kg, 3 mg/kg to 15 mg/kg, 5 mg/kg to 15 mg/kg, 10 mg/kg to 15 mg/kg, 0.5 mg/kg to 10 mg/kg, 1 mg/kg to 10 mg/kg, 2 mg/kg to 10 mg/kg, 3 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 0.5 mg/kg to 5 mg/kg, 1 mg/kg to 5 mg/kg, 2 mg/kg to 5 mg/kg, 3 mg/kg to 5 mg/kg, 0.5 mg/kg to 3 mg/kg, 1 mg/kg to 3 mg/kg, 2 mg/kg to 3 mg/kg, 0.5 mg/kg to 2 mg/kg, 1 mg/kg to 2 mg/kg, or 0.5 mg/kg to 1 mg/kg. In some embodiments, the activatable antibody inhibits the induction of type 1 diabetes in the NOD mouse after administration of the activatable antibody at a single dose of 3 mg/kg to 10 mg/kg, 3 mg/kg or 10 mg/kg. In some embodiments, the activatable antibody inhibits the induction of type 1 diabetes in the NOD mouse after administration of the activatable antibody at a single dose of 3 mg/kg to 10 mg/kg. In some embodiments, the activatable antibody inhibits the induction of type 1 diabetes in the NOD mouse after administration of the activatable antibody at a single dose of 3 mg/kg. In some embodiments, the activatable antibody inhibits the induction of type 1 diabetes in the NOD mouse after administration of the activatable antibody at a single dose of 10 mg/kg.

In some embodiments, the AB induces type 1 diabetes in the NOD mouse after administration of the AB at a single dose of 0.5 mg/kg to 15 mg/kg, 1 mg/kg to 15 mg/kg, 2 mg/kg to 15 mg/kg, 3 mg/kg to 15 mg/kg, 5 mg/kg to 15 mg/kg, 10 mg/kg to 15 mg/kg, 0.5 mg/kg to 10 mg/kg, 1 mg/kg to 10 mg/kg, 2 mg/kg to 10 mg/kg, 3 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 0.5 mg/kg to 5 mg/kg, 1 mg/kg to 5 mg/kg, 2 mg/kg to 5 mg/kg, 3 mg/kg to 5 mg/kg, 0.5 mg/kg to 3 mg/kg, 1 mg/kg to 3 mg/kg, 2 mg/kg to 3 mg/kg, 0.5 mg/kg to 2 mg/kg, 1 mg/kg to 2 mg/kg, or 0.5 mg/kg to 1 mg/kg. In some embodiments, the AB induces type 1 diabetes in the NOD mouse after administration of the AB at a single dose of 1 mg/kg to 20 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, or 20 mg/kg. In some embodiments, the AB induces type 1 diabetes in the NOD mouse after administration of the AB at a single dose of 1 mg/kg to 20 mg/kg. In some embodiments, the AB induces type 1 diabetes in the NOD mouse after administration of the AB at a single dose of 1 mg/kg. In some embodiments, the AB induces type 1 diabetes in the NOD mouse after administration of the AB at a single dose of 3 mg/kg. In some embodiments, the AB induces type 1 diabetes in the NOD mouse after administration of the AB at a single dose of 5 mg/kg. In some embodiments, the AB induces type 1 diabetes in the NOD mouse after administration of the AB at a single dose of 10 mg/kg. In some embodiments, the AB induces type 1 diabetes in the NOD mouse after administration of the AB at a single dose of 20 mg/kg.

In some embodiments, the activatable antibody has one or more of the following characteristics: (a) the activatable antibody in an uncleaved state does not induce type 1 diabetes in greater than 50% of a population of non-obese diabetic (NOD) mice, and (b) the AB induces type 1 diabetes in greater than 50%, greater than 60%, greater than 70%, greater than 80%, between 50% to 100%, between 50% and 75%, or between 70% and 90% of a population of NOD mice.

In some embodiments, the activatable antibody does not induce type 1 diabetes in greater than 50%, greater than 60%, greater than 70%, greater than 80%, between 50% to 100%, between 50% and 75%, or between 70% and 90% of the population of NOD mice after administration to each mouse in the population a single dose of the activatable antibody at a dosage of: 0.5 mg/kg to 15 mg/kg, 1 mg/kg to 15 mg/kg, 2 mg/kg to 15 mg/kg, 3 mg/kg to 15 mg/kg, 5 mg/kg to 15 mg/kg, 10 mg/kg to 15 mg/kg, 0.5 mg/kg to 10 mg/kg, 1 mg/kg to 10 mg/kg, 2 mg/kg to 10 mg/kg, 3 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 0.5 mg/kg to 5 mg/kg, 1 mg/kg to 5 mg/kg, 2 mg/kg to 5 mg/kg, 3 mg/kg to 5 mg/kg, 0.5 mg/kg to 3 mg/kg, 1 mg/kg to 3 mg/kg, 2 mg/kg to 3 mg/kg, 0.5 mg/kg to 2 mg/kg, 1 mg/kg to 2 mg/kg, or 0.5 mg/kg to 1 mg/kg. In some embodiments, the activatable antibody inhibits the induction of type 1 diabetes in the NOD mouse after administration of the activatable antibody at a single dose of 3 mg/kg to 10 mg/kg, 3 mg/kg or 10 mg/kg. In some embodiments the dose is a single dose of 3 mg/kg to 10 mg/kg. In some embodiments the dose is a single dose of 3 mg/kg. In some embodiments the dose is a single dose of 10 mg/kg.

In some embodiments, the activatable antibody has one or more of the following characteristics: (a) the activatable antibody in an uncleaved state does not induce type 1 diabetes in greater than 50%, greater than 60%, greater than 70%, greater than 80%, between 50% to 100%, between 50% and 75%, or between 70% and 90% of a population of non-obese diabetic (NOD) mice when administered at a single dose of 3 mg/kg; and (b) the AB induces type 1 diabetes in greater than 50%, greater than 60%, greater than 70%, greater than 80%, between 50% to 100%, between 50% and 75%, or between 70% and 90% of a population of NOD mice, when administered at a single dose of 3 mg/kg.

In some embodiments, the activatable antibody has one or more of the following characteristics: (a) the activatable antibody in an uncleaved state does not induce type 1 diabetes in greater than 50%, greater than 60%, greater than 70%, greater than 80%, between 50% to 100%, between 50% and 75%, or between 70% and 90% of a population of non-obese diabetic (NOD) mice when administered at a single dose of 10 mg/kg; and (b) the AB induces type 1 diabetes in greater than 50%, greater than 60%, greater than 70%, greater than 80%, between 50% to 100%, between 50% and 75%, or between 70% and 90% of a population of NOD mice, when administered at a single dose of 10 mg/kg.

In some embodiments, the AB induces type 1 diabetes in greater than 50%, greater than 60%, greater than 70%, greater than 80%, between 50% to 100%, between 50% and 75%, or between 70% and 90% of the population of the NOD mice after administration to each mouse in the population a single dose of the AB at a dosage of: 2 mg/kg to 15 mg/kg, 3 mg/kg to 15 mg/kg, 5 mg/kg to 15 mg/kg, 10 mg/kg to 15 mg/kg, 2 mg/kg to 10 mg/kg, 3 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 2 mg/kg to 5 mg/kg, 3 mg/kg to 5 mg/kg, or 2 mg/kg to 3 mg/kg. In some embodiments the dose is a single dose of 3 mg/kg to 20 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, or 20 mg/kg. In some embodiments the dose is a single dose of 3 mg/kg to 20 mg/kg. In some embodiments the dose is a single dose of 3 mg/kg. In some embodiments the dose is a single dose of 5 mg/kg. In some embodiments the dose is a single dose of 10 mg/kg. In some embodiments the dose is a single dose of 20 mg/kg.

In some embodiments, the activatable antibody in an uncleaved state does not induce type 1 diabetes in greater than 50% of a population of non-obese diabetic (NOD) mice, wherein the population of NOD mice are concurrently dosed with an anti-CTLA4 antibody.

In some embodiments, the population of NOD mice are each administered a single dose of the activatable antibody at a dosage of 2 mg/kg to 15 mg/kg, 3 mg/kg to 15 mg/kg, 5 mg/kg to 15 mg/kg, 10 mg/kg to 15 mg/kg, 2 mg/kg to 10 mg/kg, 3 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 2 mg/kg to 5 mg/kg, 3 mg/kg to 5 mg/kg, or 2 mg/kg to 3 mg/kg concurrently with a dose of an anti-CTLA4 antibody. In some embodiments, the population of NOD mice are each administered a single dose of the activatable antibody at a dosage of 10 mg/kg concurrently with a dose of an anti-CTLA4 antibody.

In some embodiments, the population of NOD mice is each administered a single dose of the anti-CTLA4 antibody at a dosage of 2 mg/kg to 15 mg/kg, 3 mg/kg to 15 mg/kg, 5 mg/kg to 15 mg/kg, or 10 mg/kg to 15 mg/kg concurrently with a dose of an anti-PD-1 activatable antibody. In some embodiments, the population of NOD mice is each administered a single dose of the anti-CTLA4 antibody at a dosage of 10 mg/kg concurrently with a dose of an anti-PD-1 activatable antibody.

In some embodiments, the population of NOD mice is each administered a single dose of the activatable antibody at a dosage of 5 mg/kg to 15 mg/kg and a single dose of the anti-CTLA4 antibody at a dosage of 5 mg/kg to 15 mg/kg. In some embodiments, the population of NOD mice is each administered a single dose of the activatable antibody at a dosage of 10 mg/kg concurrently with a single dose of an anti-CTLA4 antibody at a dosage of 10 mg/kg.

In some embodiments, the NOD mouse is a female NOD/ShiLtJ mouse substrain. In some embodiments, the population of NOD mice is each 5 weeks old when the activatable antibody is first administered. In some embodiments, the population of NOD mice is each 10 weeks old when the activatable antibody is first administered.

The activatable antibodies in an activated state bind PD-1 and include (i) an antibody or an antigen binding fragment thereof (AB) that specifically binds to PD-1; (ii) a masking moiety (MM) that, when the activatable antibody is in an uncleaved state, inhibits the binding of the AB to PD-1; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.

In some embodiments, the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM.

In some embodiments, the activatable antibody comprises a linking peptide between the MM and the CM.

In some embodiments, the activatable antibody comprises a linking peptide between the CM and the AB.

In some embodiments, the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the two linking peptides need not be identical to each other. In some embodiments, each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.

In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 363) and (GGGS)_(n) (SEQ ID NO: 364), where n is an integer of at least one.

In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 365), GGSGG (SEQ ID NO: 366), GSGSG (SEQ ID NO: 367), GSGGG (SEQ ID NO: 368), GGGSG (SEQ ID NO: 369), and GSSSG (SEQ ID NO: 370).

In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 371), GSSGGSGGSGG (SEQ ID NO: 372), GSSGGSGGSGGS (SEQ ID NO: 373), GSSGGSGGSGGSGGGS (SEQ ID NO: 374), GSSGGSGGSG (SEQ ID NO: 375), GSSGGSGGSGS (SEQ ID NO: 376), GGGSSGGS (SEQ ID NO: 65), or GGGSSGG (SEQ ID NO: 1040).

In some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 377), GSSGT (SEQ ID NO: 378) or GSSG (SEQ ID NO: 379).

In some embodiments, the AB has a dissociation constant (K_(d)) of about 100 nM or less for binding to PD-1.

In some embodiments, the activatable antibody includes an antibody or antigen-binding fragment thereof (AB) that specifically binds PD-1. In some embodiments, the antibody or antigen-binding fragment thereof that binds PD-1 is a monoclonal antibody, domain antibody, single chain, Fab fragment, a F(ab′)₂ fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody. In some embodiments, such an antibody or antigen-binding fragment thereof that binds PD-1 is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody.

In some embodiments, the activatable antibody comprises a heavy chain amino acid sequence that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37. In some embodiments, the activatable antibody comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21. In some embodiments, the activatable anti-PD-1 antibody includes a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 45 or SEQ ID NO: 47. In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21 and a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 45 or SEQ ID NO: 47. In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21 and a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 45. In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21 and a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 47.

In some embodiments, the activatable anti-PD-1 antibody includes the combination of the complementarity determining region (CDR) sequences of the heavy chain amino acid sequence of SEQ ID NO: 21. In some embodiments, the activatable anti-PD-1 antibody includes the combination of the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 45 or of SEQ ID NO: 47. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 21, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 45 or 47. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 21, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 45. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 21, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 47.

In some embodiments, the activatable anti-PD-1 antibody includes: (a) a variable heavy chain complementarity determining region 1 (VH CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 653-657; (b) a variable heavy chain complementarity determining region 2 (VH CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 658-663; (c) a variable heavy chain complementarity determining region 3 (VH CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 664-668; (d) a variable light chain complementarity determining region 1 (VL CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 669-677; (e) a variable light chain complementarity determining region 2 (VL CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 678-682; and (f) variable light chain complementarity determining region 3 (VL CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 683-687.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664).

In some embodiments, the activatable anti-PD-1 antibody includes a combination of a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675) or RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, the activatable anti-PD-1 antibody includes a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, the activatable anti-PD-1 antibody includes a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VL CDR1 sequence comprises RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683).

In some embodiments, the activatable anti-PD-1 antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664); the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675) or RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, the activatable anti-PD-1 antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664); the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, the activatable anti-PD-1 antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664); the VL CDR1 sequence comprises RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683).

In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 1346, and a light chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 626.

In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 1514, and a light chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 638.

In some embodiments, the activatable anti-PD-1 antibody includes the combination of the complementarity determining region (CDR) sequences of the heavy chain amino acid sequence of SEQ ID NO: 1346. In some embodiments, the activatable anti-PD-1 antibody includes the combination of the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 626. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 1346, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 626.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, wherein the VH CDR1 sequence comprises GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises IWYDGSKR (SEQ ID NO: 1706); and the VH CDR3 sequence comprises TNDDY (SEQ ID NO: 1707).

In some embodiments, the activatable anti-PD-1 antibody includes a combination of a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein the VL CDR1 sequence comprises QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises QQSSNWPRT (SEQ ID NO: 1710).

In some embodiments, the activatable anti-PD-1 antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises IWYDGSKR (SEQ ID NO: 1706); the VH CDR3 sequence comprises TNDDY (SEQ ID NO: 1707); the VL CDR1 sequence comprises QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises QQSSNWPRT (SEQ ID NO: 1710).

In some embodiments, the activatable anti-PD-1 antibody includes the combination of the complementarity determining region (CDR) sequences of the heavy chain amino acid sequence of SEQ ID NO: 1514. In some embodiments, the activatable anti-PD-1 antibody includes the combination of the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 638. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 1514, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 638.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, wherein the VH CDR1 sequence comprises GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises INPSNGGT (SEQ ID NO: 1712); and the VH CDR3 sequence comprises RRDYRFDMGFDY (SEQ ID NO: 1713).

In some embodiments, the activatable anti-PD-1 antibody includes a combination of a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein the VL CDR1 sequence comprises KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises QHSRDLPLT (SEQ ID NO: 1716).

In some embodiments, the activatable anti-PD-1 antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises INPSNGGT (SEQ ID NO: 1712); the VH CDR3 sequence comprises RRDYRFDMGFDY (SEQ ID NO: 1713); the VL CDR1 sequence comprises KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises QHSRDLPLT (SEQ ID NO: 1716).

In some embodiments, the activatable antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37. In some embodiments, the activatable antibody comprises a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, the activatable antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the activatable antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37. In some embodiments, the activatable antibody comprises a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, the activatable antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21, and a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 47. In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21, and a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21, and a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 47.

In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1346, and a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 626.

In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1514, and a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 638.

In some embodiments, the AB of the activatable anti-PD-1 antibody comprises a heavy chain amino acid sequence selected from the group consisting of the heavy chain sequences shown in Table 7. In some embodiments, the AB of the activatable anti-PD-1 antibody comprises a light chain amino acid sequence selected from the group consisting of the light chain sequences shown in Table 7. In some embodiments, the AB of the activatable anti-PD-1 antibody comprises a heavy chain amino acid sequence selected from the group consisting of the heavy chain sequences shown in Table 7 and a light chain amino acid sequence selected from the group consisting of the light chain sequences shown in Table 7.

In some embodiments, the AB of the activatable anti-PD-1 antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of the heavy chain sequences shown in Table 7. In some embodiments, the AB of the activatable anti-PD-1 antibody comprises a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of the light chain sequences shown in Table 7. In some embodiments, the AB of the activatable anti-PD-1 antibody comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of the heavy chain sequences shown in Table 7 and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of the light chain sequences shown in Table 7.

In some embodiments, the AB of the activatable antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to IWYDGSKR (SEQ ID NO: 1706); and the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TNDDY (SEQ ID NO: 1707).

In some embodiments, the AB of the activatable antibody includes a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QQSSNWPRT (SEQ ID NO: 1710).

In some embodiments, the AB of the activatable antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to IWYDGSKR (SEQ ID NO: 1706); the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to TNDDY (SEQ ID NO: 1707); the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QQSSNWPRT (SEQ ID NO: 1710).

In some embodiments, the AB of the activatable antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to INPSNGGT (SEQ ID NO: 1712); and the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to RRDYRFDMGFDY (SEQ ID NO: 1713).

In some embodiments, the AB of the activatable antibody includes a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QHSRDLPLT (SEQ ID NO: 1716).

In some embodiments, the AB of the activatable antibody includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to INPSNGGT (SEQ ID NO: 1712); the VH CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to RRDYRFDMGFDY (SEQ ID NO: 1713); the VL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to QHSRDLPLT (SEQ ID NO: 1716).

In some embodiments, the activatable antibody comprises a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence shown in Table 8; a VH CDR2 sequence shown in Table 8; a VH CDR3 sequence shown in Table 8; a VL CDR1 sequence shown in Table 8; a VL CDR2 sequence shown in Table 8; and a VL CDR3 sequence shown in Table 8.

In some embodiments, the activatable antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence shown in Table 8; a VH CD2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence shown in Table 8; a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR3 sequence shown in Table 8; a VL CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR1 sequence shown in Table 8; a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence shown in Table 8; and a VL CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR3 sequence shown in Table 8.

In some embodiments, the activatable antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence shown in a single row in Table 8, and a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence shown in a single row in Table 8.

In some embodiments, the antibody comprises a heavy chain that comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the combination is a combination of the three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single row in Table 8.

In some embodiments, the activatable antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence shown in a single row in Table 8, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence shown in a single row in Table 8, wherein each CDR sequence in the combination comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the corresponding CDR sequence in a combination of the three VH CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single row in Table 8 and in a combination of the three VL CDR sequences (VL CDR1, VL CDR2, and VL CDR3) shown in a single row in Table 8.

In some embodiments, the antibody comprises a heavy chain that comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein each CDR sequence in the combination comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the corresponding CDR sequence in a combination of three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single row in Table 8.

In some embodiments, the activatable antibody comprises a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence shown in Table 9; a VH CDR2 sequence shown in Table 9; a VH CDR3 sequence shown in Table 9; a VL CDR1 sequence shown in Table 9; a VL CDR2 sequence shown in Table 9; and a VL CDR3 sequence shown in Table 9.

In some embodiments, the activatable antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence shown in Table 9; a VH CD2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence shown in Table 9; a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR3 sequence shown in Table 9; a VL CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR1 sequence shown in Table 9; a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence shown in Table 9; and a VL CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR3 sequence shown in Table 9.

In some embodiments, the activatable antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination is a combination shown in Table 9.

In some embodiments, the activatable antibody comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein each CDR sequence in the combination comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the corresponding CDR sequence in a combination shown in Table 9.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 675 or SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a cleavable moiety; a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 675 or SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664, and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 675 or SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664, and a human immunoglobulin heavy chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of 381, 382, 383, and 1807.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 675 or SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664, a human immunoglobulin heavy chain constant region, and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807, and the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

n some embodiments, the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 675; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 675; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664, and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 675; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664, and a human immunoglobulin heavy chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 675; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664, a human immunoglobulin heavy chain constant region, and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807, and the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664, and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664, and a human immunoglobulin heavy chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664, a human immunoglobulin heavy chain constant region, and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807, and the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 21 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, 99; a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; and a combination of a variable heavy chain (VH) sequence of SEQ ID NO: 21 and a variable light chain (VL) sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), which is referred to herein as mask PD034 or PD34, a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDNP (SEQ ID NO: 1100), which is also referred to herein as substrate 2011, and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 21 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDNP (SEQ ID NO: 1100), and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDNP (SEQ ID NO: 1100), and a combination of a variable heavy chain (VH) sequence of SEQ ID NO: 21 and a variable light chain (VL) sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), which is referred to herein as mask PD034 or PD34, a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSANP (SEQ ID NO: 1101), which is also referred to herein as substrate 2012, and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 21 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSANP (SEQ ID NO: 1101), and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSANP (SEQ ID NO: 1101), and a combination of a variable heavy chain (VH) sequence of SEQ ID NO: 21 and a variable light chain (VL) sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), which is referred to herein as mask PD034 or PD34, a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDNP (SEQ ID NO: 1100), which is also referred to herein as substrate 2011, and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 21 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDNP (SEQ ID NO: 1100), and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDNP (SEQ ID NO: 1100), and a combination of a variable heavy chain (VH) sequence of SEQ ID NO: 21 and a variable light chain (VL) sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), which is referred to herein as mask PD034 or PD34, a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDNH (SEQ ID NO: 214), which is also referred to herein as substrate 2001, and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 21 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDNH (SEQ ID NO: 214), and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDNH (SEQ ID NO: 214), and a combination of a variable heavy chain (VH) sequence of SEQ ID NO: 21 and a variable light chain (VL) sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSGNH (SEQ ID NO: 361), which is also referred to herein as substrate 2002, and a combination selected from the group consisting of (a) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 21 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 47; (b) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664; and (c) a combination of a variable heavy chain (VH) sequence of SEQ ID NO: 21 and a variable light chain (VL) sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSANPRG (SEQ ID NO: 1092), which is also referred to herein as substrate 2003, and a combination selected from the group consisting of (a) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 21 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 47; (b) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664; and (c) a combination of a variable heavy chain (VH) sequence of SEQ ID NO: 21 and a variable light chain (VL) sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDDH (SEQ ID NO: 1095), which is also referred to herein as substrate 2006, and a combination selected from the group consisting of (a) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 21 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 47; (b) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664; and (c) a combination of a variable heavy chain (VH) sequence of SEQ ID NO: 21 and a variable light chain (VL) sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDIH (SEQ ID NO: 1096), which is also referred to herein as substrate 2007, and a combination selected from the group consisting of (a) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 21 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 47; (b) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664; and (c) a combination of a variable heavy chain (VH) sequence of SEQ ID NO: 21 and a variable light chain (VL) sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDQH (SEQ ID NO: 1097), which is also referred to herein as 2008, and a combination selected from the group consisting of (a) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 21 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 47; (b) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664; and (c) a combination of a variable heavy chain (VH) sequence of SEQ ID NO: 21 and a variable light chain (VL) sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDTH (SEQ ID NO: 1098), which is also referred to herein as substrate 2009, and a combination selected from the group consisting of (a) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 21 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 47; (b) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664; and (c) a combination of a variable heavy chain (VH) sequence of SEQ ID NO: 21 and a variable light chain (VL) sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDYH (SEQ ID NO: 1099), which is also referred to herein as 2010, and a combination selected from the group consisting of (a) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 21 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 47; (b) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664; and (c) a combination of a variable heavy chain (VH) sequence of SEQ ID NO: 21 and a variable light chain (VL) sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSANI (SEQ ID NO: 1102), which is also referred to herein as substrate 2013, and a combination selected from the group consisting of (a) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 21 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 47; (b) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664; and (c) a combination of a variable heavy chain (VH) sequence of SEQ ID NO: 21 and a variable light chain (VL) sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the amino acid sequence TSYCSIEHYPCNTHH (SEQ ID NO: 99), a cleavable moiety (CM) comprising the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 1111), which is also referred to herein as substrate 2014, and a combination selected from the group consisting of (a) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 21 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 47; (b) a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VL CDR1 sequence of SEQ ID NO: 676; the VL CDR2 sequence of SEQ ID NO: 678; the VL CDR3 sequence of SEQ ID NO: 683; the VH CDR1 sequence of SEQ ID NO: 653; the VH CDR2 sequence of SEQ ID NO: 658; and the VH CDR3 sequence of SEQ ID NO: 664; and (c) a combination of a variable heavy chain (VH) sequence of SEQ ID NO: 21 and a variable light chain (VL) sequence of SEQ ID NO: 47. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1151; and a heavy chain comprising the amino acid sequence of SEQ ID NO:21.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1152; and a heavy chain comprising the amino acid sequence of SEQ ID NO:21.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1190; and a heavy chain comprising the amino acid sequence of SEQ ID NO:21.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1191; and a heavy chain comprising the amino acid sequence of SEQ ID NO:21.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1198; and a heavy chain comprising the amino acid sequence of SEQ ID NO:21.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1199; and a heavy chain comprising the amino acid sequence of SEQ ID NO:21.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2055; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2052.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2054; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2052.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2057; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2052.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2056; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2052.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2059; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2052.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2058; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2052.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2055; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2053.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2054; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2053.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2057; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2053.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2056; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2053.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2059; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2053.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2058; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2053.

In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1351-1362; a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 1514 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 638. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1351-1362; a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises INPSNGGT (SEQ ID NO: 1712); the VH CDR3 sequence comprises RRDYRFDMGFDY (SEQ ID NO: 1713); the VL CDR1 sequence comprises KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises QHSRDLPLT (SEQ ID NO: 1716). In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1351-1362; a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; the VH sequence of SEQ ID NO: 1514 and the VL sequence of SEQ ID NO: 638. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1206-1217; a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination comprises the VH CDR sequences of the variable heavy chain sequence of SEQ ID NO: 1346 and the VL CDR sequences of the variable light chain sequence of SEQ ID NO: 626. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1206-1217; a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; and a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises IWYDGSKR (SEQ ID NO: 1706); the VH CDR3 sequence comprises TNDDY (SEQ ID NO: 1707); the VL CDR1 sequence comprises QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises QQSSNWPRT (SEQ ID NO: 1710). In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a MM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1206-1217; a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157; the VH sequence of SEQ ID NO: 1346 and the VL sequence of SEQ ID NO: 626. In some embodiments, the combination further comprises a human immunoglobulin heavy chain constant region, a human immunoglobulin light chain constant domain, or both a human immunoglobulin heavy chain constant region and a human immunoglobulin light chain constant domain. In some embodiments, the human immunoglobulin heavy chain constant domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 381, 382, 383, and 1807. In some embodiments, the human immunoglobulin light chain constant domain is a human kappa constant domain comprising SEQ ID NO: 61 or SEQ ID NO: 1344.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSANP (SEQ ID NO: 1101), which is also referred to herein as substrate 2012; the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDNP (SEQ ID NO: 1100), which is also referred to herein as substrate 2011; the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDNH (SEQ ID NO: 214), which is also referred to herein as substrate 2001; the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSGNH (SEQ ID NO: 361), which is also referred to herein as substrate 2002; the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSANPRG (SEQ ID NO: 1092), which is also referred to herein as substrate 2003; the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDDH (SEQ ID NO: 1095), which is also referred to herein as substrate 2006; the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDIH (SEQ ID NO: 1096), which is also referred to herein as substrate 2007; the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDQH (SEQ ID NO: 1097), which is also referred to herein as substrate 2008; the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDTH (SEQ ID NO: 1098), which is also referred to herein as substrate 2009; the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDQH (SEQ ID NO: 1099), which is also referred to herein as substrate 2010; the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSANI (SEQ ID NO: 1102), which is also referred to herein as substrate 2013; the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 1111), which is also referred to herein as substrate 2014; the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSANP (SEQ ID NO: 1101), which is also referred to herein as substrate 2012; the variable heavy chain region (VH) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the variable light chain region (VL) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDNP (SEQ ID NO: 1100), which is also referred to herein as substrate 2011; the variable heavy chain region (VH) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the variable light chain region (VL) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDNH (SEQ ID NO: 214), which is also referred to herein as substrate 2001; the variable heavy chain region (VH) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the variable light chain region (VL) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSGNH (SEQ ID NO: 361), which is also referred to herein as substrate 2002; the variable heavy chain region (VH) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the variable light chain region (VL) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSANPRG (SEQ ID NO: 1092), which is also referred to herein as substrate 2003; the variable heavy chain region (VH) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the variable light chain region (VL) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDDH (SEQ ID NO: 1095), which is also referred to herein as substrate 2006; the variable heavy chain region (VH) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the variable light chain region (VL) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDIH (SEQ ID NO: 1096), which is also referred to herein as substrate 2007; the variable heavy chain region (VH) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the variable light chain region (VL) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDQH (SEQ ID NO: 1097), which is also referred to herein as substrate 2008; the variable heavy chain region (VH) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the variable light chain region (VL) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDTH (SEQ ID NO: 1098), which is also referred to herein as substrate 2009; the variable heavy chain region (VH) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the variable light chain region (VL) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDYH (SEQ ID NO: 1099), which is also referred to herein as substrate 2010; the variable heavy chain region (VH) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the variable light chain region (VL) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSANI (SEQ ID NO: 1102), which is also referred to herein as substrate 2013; the variable heavy chain region (VH) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the variable light chain region (VL) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 1111), which is also referred to herein as substrate 2014; the variable heavy chain region (VH) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the variable light chain region (VL) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSANP (SEQ ID NO: 1101), which is also referred to herein as substrate 2012; the heavy chain complementarity determining regions (CDRs) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain complementarity determining regions (CDRs) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDNP (SEQ ID NO: 1100), which is also referred to herein as substrate 2011; the heavy chain complementarity determining regions (CDRs) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain complementarity determining regions (CDRs) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDNH (SEQ ID NO: 214), which is also referred to herein as substrate 2001; the heavy chain complementarity determining regions (CDRs) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain complementarity determining regions (CDRs) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSGNH (SEQ ID NO: 361), which is also referred to herein as substrate 2002; the heavy chain complementarity determining regions (CDRs) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain complementarity determining regions (CDRs) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSANPRG (SEQ ID NO: 1092), which is also referred to herein as substrate 2003; the heavy chain complementarity determining regions (CDRs) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain complementarity determining regions (CDRs) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDDH (SEQ ID NO: 1095), which is also referred to herein as substrate 2006; the heavy chain complementarity determining regions (CDRs) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain complementarity determining regions (CDRs) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDIH (SEQ ID NO: 1096), which is also referred to herein as substrate 2007; the heavy chain complementarity determining regions (CDRs) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain complementarity determining regions (CDRs) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDQH (SEQ ID NO: 1097), which is also referred to herein as substrate 2008; the heavy chain complementarity determining regions (CDRs) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain complementarity determining regions (CDRs) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDTH (SEQ ID NO: 1098), which is also referred to herein as substrate 2009; the heavy chain complementarity determining regions (CDRs) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain complementarity determining regions (CDRs) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDYH (SEQ ID NO: 1099), which is also referred to herein as substrate 2010; the heavy chain complementarity determining regions (CDRs) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain complementarity determining regions (CDRs) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSANI (SEQ ID NO: 1102), which is also referred to herein as substrate 2013; the heavy chain complementarity determining regions (CDRs) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain complementarity determining regions (CDRs) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a MM comprising the amino acid sequence ACRICQDHPATKWNS (SEQ ID NO: 549), a CM comprising the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 1111), which is also referred to herein as substrate 2014; the heavy chain complementarity determining regions (CDRs) of the heavy chain (HC) sequence of SEQ ID NO: 546, and the light chain complementarity determining regions (CDRs) of the light chain (LC) sequence of SEQ ID NO: 543.

In some embodiments, the activatable antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828, and 1830; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 546.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1808; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 546.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1810; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 546.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1812; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 546.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1814; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 546.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1816; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 546.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1818; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 546.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1820; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 546.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1822; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 546.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1824; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 546.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1826; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 546.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1828; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 546.

In some embodiments, the activatable antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1830; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 546.

In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37. In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37.

In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37. In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37.

In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a nucleic acid sequence encoding a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that comprises a nucleic acid sequence encoding a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a nucleic acid sequence that comprises a nucleic acid sequence encoding a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, the activatable antibody is encoded by a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 18, 22, 24, 26, 28, 30, 32, 34, 36, and 38. In some embodiments, the activatable antibody is encoded by a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 36, and 38.

In some embodiments, the activatable antibody is encoded by a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, 8, 12, 16, 20, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60. In some embodiments, the activatable antibody is encoded by a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60.

In some embodiments, the activatable antibody is encoded by heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: selected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 18, 22, 24, 26, 28, 30, 32, 34, 36, and 38, and a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, 8, 12, 16, 20, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60. In some embodiments, the activatable antibody is encoded by heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: selected from the group consisting of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 36, and 38, and a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60.

In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 18, 22, 24, 26, 28, 30, 32, 34, 36, and 38. In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 36, and 38.

In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, 8, 12, 16, 20, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60. In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60.

In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 18, 22, 24, 26, 28, 30, 32, 34, 36, and 38, and a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, 8, 12, 16, 20, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60. In some embodiments, the activatable antibody is encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a heavy chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 36, and 38, and a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a light chain nucleic acid sequence selected from the group consisting of SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60.

In some embodiments, the MM has a dissociation constant, i.e., dissociation constant at an equilibrium state, K_(d) for binding to the AB that is greater than the K_(d) for binding of the AB to PD-1.

In some embodiments, the MM has a K_(d) for binding to the AB that is no more than the K_(d) for binding of the AB to PD-1.

In some embodiments, the MM has a K_(d) for binding to the AB that is no less than the K_(d) for binding of the AB to PD-1.

In some embodiments, the MM has a K_(d) for binding to the AB that is approximately equal to the K_(d) for binding of the AB to PD-1.

In some embodiments, the MM has a K_(d) for binding to the AB that is less than the K_(d) for binding of the AB to PD-1.

In some embodiments, the MM has a K_(d) for binding to the AB that is no more than 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 fold greater than the K_(d) for binding of the AB to PD-1. In some embodiments, the MM has a K_(d) for binding to the AB that is between 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1000, or 100-1,000 fold greater than the K_(d) for binding of the AB to PD-1.

In some embodiments, the MM has an affinity for binding to the AB that is less than the affinity of binding of the AB to PD-1.

In some embodiments, the MM has an affinity for binding to the AB that is no more than the affinity of binding of the AB to PD-1.

In some embodiments, the MM has an affinity for binding to the AB that is approximately equal of the affinity of binding of the AB to PD-1.

In some embodiments, the MM has an affinity for binding to the AB that is no less than the affinity of binding of the AB to PD-1.

In some embodiments, the MM has an affinity for binding to the AB that is greater than the affinity of binding of the AB to PD-1.

In some embodiments, the MM has an affinity for binding to the AB that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 less than the affinity of binding of the AB to PD-1. I In some embodiments, the MM has an affinity for binding to the AB that is between 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1000, or 100-1,000 fold less than the affinity of binding of the AB to PD-1. In some embodiments, the MM has an affinity for binding to the AB that is 2 to 20 fold less than the affinity of binding of the AB to PD-1. In some embodiments, a MM not covalently linked to the AB and at equimolar concentration to the AB does not inhibit the binding of the AB to PD-1.

In some embodiments, the MM does not interfere or compete with the AB for binding to PD-1 when the activatable antibody is in a cleaved state.

In some embodiments, the MM is a polypeptide of about 2 to 40 amino acids in length. In some embodiments, the MM is a polypeptide of up to about 40 amino acids in length.

In some embodiments, the MM is a polypeptide of no more than 40 amino acids in length.

In some embodiments, the MM polypeptide sequence is different from that of PD-1. In some embodiments, the MM polypeptide sequence is different from that of human PD-1. In some embodiments, the MM polypeptide sequence is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM polypeptide sequence is different from that of PD-1 and is no more than 40%, 30%, 25%, 20%, 15%, or 10% identical to any natural binding partner of the AB. In some embodiments, the MM polypeptide sequence is no more than 50% identical to human PD-1. In some embodiments, the MM polypeptide sequence is different from that of PD-1 and is no more than 40%, 30%, 25%, 20%, 15%, or 10% identical to human PD-1.

In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-213, 384-514, 548-571, 1206-1295, and 1351-1465. In some embodiments, the MM comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-213, 384-514, 548-571, 1206-1295, and 1351-1465. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 71, 74, 77, 81, 82, 84, 90, 91, 93, and 99. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 71, 74, 77, 82, 84, 90, 91, 93, and 99. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1206-1217. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1351-1362.

In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-213. In some embodiments, the MM comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-213.

In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 384-514. In some embodiments, the MM comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 384-514.

In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 548-571. In some embodiments, the MM comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 548-571.

In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1206-1295. In some embodiments, the MM comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1206-1295.

In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1351-1465. In some embodiments, the MM comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1351-1465.

In some embodiments, the antibody or activatable antibody comprises a heavy chain comprising an amino acid selected from the group consisting of SEQ ID NOs: 2052 and 2053.

In some embodiments, the antibody or activatable antibody comprises a light chain comprising an amino acid selected from the group consisting of SEQ ID NOs: 2054 to 2059.

In some embodiments, the antibody or activatable antibody comprises a heavy chain comprising an amino acid selected from the group consisting of SEQ ID NOs: 2052 and 2053 and a light chain comprising an amino acid selected from the group consisting of SEQ ID NOs: 2054 to 2059.

In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind PD-1 such that the dissociation constant (K_(d)) of the AB when coupled to the MM towards PD-1 is at least two times greater than the K_(d) of the AB when not coupled to the MM towards PD-1.

In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind PD-1 such that the dissociation constant (K_(d)) of the AB when coupled to the MM towards PD-1 is at least five times greater than the K_(d) of the AB when not coupled to the MM towards PD-1.

In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind PD-1 such that the dissociation constant (K_(d)) of the AB when coupled to the MM towards PD-1 is at least 10 times greater than the K_(d) of the AB when not coupled to the MM towards PD-1.

In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind PD-1 such that the dissociation constant (K_(d)) of the AB when coupled to the MM towards PD-1 is at least 20 times greater than the K_(d) of the AB when not coupled to the MM towards PD-1.

In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind PD-1 such that the dissociation constant (K_(d)) of the AB when coupled to the MM towards PD-1 is at least 40 times greater than the K_(d) of the AB when not coupled to the MM towards PD-1.

In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind PD-1 such that the dissociation constant (K_(d)) of the AB when coupled to the MM towards PD-1 is at least 100 times greater than the K_(d) of the AB when not coupled to the MM towards PD-1.

In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind PD-1 such that the dissociation constant (K_(d)) of the AB when coupled to the MM towards PD-1 is at least 1000 times greater than the K_(d) of the AB when not coupled to the MM towards PD-1.

In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind PD-1 such that the dissociation constant (K_(d)) of the AB when coupled to the MM towards PD-1 is at least 10,000 times greater than the K_(d) of the AB when not coupled to the MM towards PD-1.

In some embodiments, in the presence of PD-1, the MM reduces the ability of the AB to bind PD-1 by at least 90% when the CM is uncleaved, as compared to when the CM is cleaved when assayed in vitro using a target displacement assay such as, for example, the assay described in PCT Publication No. WO 2010/081173, the contents of which are hereby incorporated by reference in their entirety.

In some embodiments, the protease that cleaves the CM is active, e.g., up-regulated, in diseased tissue, and the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease.

In some embodiments, the protease is produced by a tumor that is in proximity to T cells that express PD-1, and the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease. In some embodiments, the protease is produced by a tumor that is co-localized with T cells that express PD-1, and the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease.

In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to PD-1 is reduced to occur with a dissociation constant that is at least twofold greater than the dissociation constant of an unmodified AB binding to PD-1, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds PD-1.

In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to PD-1 is reduced to occur with a dissociation constant that is at least fivefold greater than the dissociation constant of an unmodified AB binding to PD-1, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds PD-1.

In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to PD-1 is reduced to occur with a dissociation constant that is at least 10-fold greater than the dissociation constant of an unmodified AB binding to PD-1, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds PD-1.

In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to PD-1 is reduced to occur with a dissociation constant that is at least 20-fold greater than the dissociation constant of an unmodified AB binding to PD-1, whereas in the cleaved state (i.e., when the activatable antibody is in the cleaved state), the AB binds PD-1.

In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to PD-1 is reduced to occur with a dissociation constant that is at least 40-fold greater than the dissociation constant of an unmodified AB binding to PD-1, whereas in the cleaved state, the AB binds PD-1.

In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to PD-1 is reduced to occur with a dissociation constant that is at least 50-fold greater than the dissociation constant of an unmodified AB binding to PD-1, whereas in the cleaved state, the AB binds PD-1.

In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to PD-1 is reduced to occur with a dissociation constant that is at least 100-fold greater than the dissociation constant of an unmodified AB binding to PD-1, whereas in the cleaved state, the AB binds PD-1.

In some embodiments, the CM is positioned in the activatable antibody such that when the activatable antibody is in the uncleaved state, binding of the activatable antibody to PD-1 is reduced to occur with a dissociation constant that is at least 200-fold greater than the dissociation constant of an unmodified AB binding to PD-1, whereas in the cleaved state, the AB binds PD-1.

In some embodiments, the CM is a polypeptide of up to 15 amino acids in length.

In some embodiments, the CM is a polypeptide that includes a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP). In some embodiments, each of the CM1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate is independently a polypeptide of up to 15 amino acids in length.

In some embodiments, the CM is a substrate for at least one protease that is or is believed to be up-regulated in cancer. In some embodiments, the CM is a substrate for at least one protease that is or is believed to be up-regulated in inflammation. In some embodiments, the CM is a substrate for at least one protease that is or is believed to be up-regulated in autoimmunity.

In some embodiments, the CM is a substrate for at least one protease selected from the group consisting of a matrix metalloprotease (MMP), thrombin, a neutrophil elastase, a cysteine protease, legumain, and a serine protease, such as matriptase (MT-SP1), and urokinase (uPA). Without being bound by theory, it is believed that these proteases are up-regulated in at least one of cancer, inflammation, and/or autoimmunity.

Exemplary substrates include but are not limited to substrates cleavable by one or more of the following enzymes or proteases listed in Table 3.

In some embodiments, the CM is selected for use with a specific protease, for example a protease that is known to be co-localized with the target of the activatable antibody. For example, the protease is produced by a tumor that is in proximity to T cells that express PD-1. In some embodiments, the protease is produced by a tumor that is co-localized with T cells that express PD-1.

In some embodiments, the CM is a substrate for at least one MMP. Examples of MMPs include the MMPs listed in the Table 3. In some embodiments, the CM is a substrate for a protease selected from the group consisting of MMP 9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. In some embodiments the CM is a substrate for MMP9. In some embodiments, the CM is a substrate for MMP14.

In some embodiments, the CM is a substrate that includes the sequence TGRGPSWV (SEQ ID NO: 295); SARGPSRW (SEQ ID NO: 319); TARGPSFK (SEQ ID NO: 297); LSGRSDNH (SEQ ID NO: 294); GGWHTGRN (SEQ ID NO: 320); HTGRSGAL (SEQ ID NO: 321); PLTGRSGG (SEQ ID NO: 296); AARGPAIH (SEQ ID NO: 322); RGPAFNPM (SEQ ID NO: 323); SSRGPAYL (SEQ ID NO: 324); RGPATPIM (SEQ ID NO: 325); RGPA (SEQ ID NO: 326); GGQPSGMWGW (SEQ ID NO: 327); FPRPLGITGL (SEQ ID NO: 328); VHMPLGFLGP (SEQ ID NO: 302); SPLTGRSG (SEQ ID NO: 329); SAGFSLPA (SEQ ID NO: 330); LAPLGLQRR (SEQ ID NO: 331); SGGPLGVR (SEQ ID NO: 332); PLGL (SEQ ID NO: 333); LSGRSGNH (SEQ ID NO: 1157); SGRSANPRG (SEQ ID NO: 1158); LSGRSDDH (SEQ ID NO: 1161); LSGRSDIH (SEQ ID NO: 1162); LSGRSDQH (SEQ ID NO: 1165); LSGRSDTH (SEQ ID NO: 1166); LSGRSDDH (SEQ ID NO: 1169); LSGRSDNP (SEQ ID NO: 1520); LSGRSANP (SEQ ID NO: 1695); LSGRSANI (SEQ ID NO: 1696); and/or LSGRSDNI (SEQ ID NO: 1697).

In some embodiments, the CM comprises the amino acid sequence LSGRSDNH (SEQ ID NO: 294). In some embodiments, the CM comprises the amino acid sequence TGRGPSWV (SEQ ID NO: 295). In some embodiments, the CM comprises the amino acid sequence PLTGRSGG (SEQ ID NO: 296). In some embodiments, the CM comprises the amino acid sequence GGQPSGMWGW (SEQ ID NO: 327). In some embodiments, the CM comprises the amino acid sequence FPRPLGITGL (SEQ ID NO: 328). In some embodiments, the CM comprises the amino acid sequence VHMPLGFLGP (SEQ ID NO: 302). In some embodiments, the CM comprises the amino acid sequence PLGL (SEQ ID NO: 333). In some embodiments, the CM comprises the amino acid sequence SARGPSRW (SEQ ID NO: 319). In some embodiments, the CM comprises the amino acid sequence TARGPSFK (SEQ ID NO: 297). In some embodiments, the CM comprises the amino acid sequence GGWHTGRN (SEQ ID NO: 320). In some embodiments, the CM comprises the amino acid sequence HTGRSGAL (SEQ ID NO: 321). In some embodiments, the CM comprises the amino acid sequence AARGPAIH (SEQ ID NO: 322). In some embodiments, the CM comprises the amino acid sequence RGPAFNPM (SEQ ID NO: 323). In some embodiments, the CM comprises the amino acid sequence SSRGPAYL (SEQ ID NO: 324). In some embodiments, the CM comprises the amino acid sequence RGPATPIM (SEQ ID NO: 325). In some embodiments, the CM comprises the amino acid sequence RGPA (SEQ ID NO: 326). In some embodiments, the CM comprises the amino acid sequence LSGRSGNH (SEQ ID NO: 1157). In some embodiments, the CM comprises the amino acid sequence SGRSANPRG (SEQ ID NO: 1158). In some embodiments, the CM comprises the amino acid sequence LSGRSDDH (SEQ ID NO: 1161). In some embodiments, the CM comprises the amino acid sequence LSGRSDIH (SEQ ID NO: 1162). In some embodiments, the CM comprises the amino acid sequence LSGRSDQH (SEQ ID NO: 1165). In some embodiments, the CM comprises the amino acid sequence LSGRSDTH (SEQ ID NO: 1166). In some embodiments, the CM comprises the amino acid sequence LSGRSDYH (SEQ ID NO: 1169). In some embodiments, the CM comprises the amino acid sequence LSGRSDNP (SEQ ID NO: 1520). In some embodiments, the CM comprises the amino acid sequence LSGRSANP (SEQ ID NO: 1695). In some embodiments, the CM comprises the amino acid sequence LSGRSANI (SEQ ID NO: 1696). In some embodiments, the CM comprises the amino acid sequence LSGRSDNI (SEQ ID NO: 1697).

In some embodiments, the CM is a substrate for an MMP and includes the sequence ISSGLSS (SEQ ID NO: 334); QNQALRMA (SEQ ID NO: 305); AQNLLGMV (SEQ ID NO: 304); STFPFGMF (SEQ ID NO: 307); PVGYTSSL (SEQ ID NO: 335); DWLYWPGI (SEQ ID NO: 336), ISSGLLSS (SEQ ID NO: 308), LKAAPRWA (SEQ ID NO: 337); GPSHLVLT (SEQ ID NO: 338); LPGGLSPW (SEQ ID NO: 339); MGLFSEAG (SEQ ID NO: 340); SPLPLRVP (SEQ ID NO: 341); RMHLRSLG (SEQ ID NO: 342); LAAPLGLL (SEQ ID NO: 306); AVGLLAPP (SEQ ID NO: 303); LLAPSHRA (SEQ ID NO: 343); PAGLWLDP (SEQ ID NO: 309); MIAPVAYR (SEQ ID NO: 1698); RPSPMWAY (SEQ ID NO: 1699); WATPRPMR (SEQ ID NO: 1700); FRLLDWQW (SEQ ID NO: 1701); ISSGL (SEQ ID NO: 1702); ISSGLLS (SEQ ID NO: 1703); and/or ISSGLL (SEQ ID NO: 1704).

In some embodiments, the CM comprises the amino acid sequence ISSGLSS (SEQ ID NO: 334). In some embodiments, the CM comprises the amino acid sequence QNQALRMA (SEQ ID NO: 305). In some embodiments, the CM comprises the amino acid sequence AQNLLGMV (SEQ ID NO: 304). In some embodiments, the CM comprises the amino acid sequence STFPFGMF (SEQ ID NO: 307). In some embodiments, the CM comprises the amino acid sequence PVGYTSSL (SEQ ID NO: 335). In some embodiments, the CM comprises the amino acid sequence DWLYWPGI (SEQ ID NO: 336). In some embodiments, the CM comprises the amino acid sequence ISSGLLSS (SEQ ID NO: 308). In some embodiments, the CM comprises the amino acid sequence LKAAPRWA (SEQ ID NO: 337). In some embodiments, the CM comprises the amino acid sequence GPSHLVLT (SEQ ID NO: 338). In some embodiments, the CM comprises the amino acid sequence LPGGLSPW (SEQ ID NO: 339). In some embodiments, the CM comprises the amino acid sequence MGLFSEAG (SEQ ID NO: 340). In some embodiments, the CM comprises the amino acid sequence SPLPLRVP (SEQ ID NO: 341). In some embodiments, the CM comprises the amino acid sequence RMHLRSLG (SEQ ID NO: 342). In some embodiments, the CM comprises the amino acid sequence LAAPLGLL (SEQ ID NO: 306). In some embodiments, the CM comprises the amino acid sequence AVGLLAPP (SEQ ID NO: 303). In some embodiments, the CM comprises the amino acid sequence LLAPSHRA (SEQ ID NO: 343). In some embodiments, the CM comprises the amino acid sequence PAGLWLDP (SEQ ID NO: 309). In some embodiments, the CM comprises the amino acid sequence MIAPVAYR (SEQ ID NO: 1698). In some embodiments, the CM comprises the amino acid sequence RPSPMWAY (SEQ ID NO: 1699). In some embodiments, the CM comprises the amino acid sequence WATPRPMR (SEQ ID NO: 1700). In some embodiments, the CM comprises the amino acid sequence FRLLDWQW (SEQ ID NO: 1701). In some embodiments, the CM comprises the amino acid sequence ISSGL (SEQ ID NO: 1702). In some embodiments, the CM comprises the amino acid sequence ISSGLLS (SEQ ID NO: 1703). In some embodiments, the CM comprises the amino acid sequence ISSGLL (SEQ ID NO: 1704).

In some embodiments, the CM is a substrate for thrombin. In some embodiments, the CM is a substrate for thrombin and includes the sequence GPRSFGL (SEQ ID NO: 344) or GPRSFG (SEQ ID NO: 345). In some embodiments, the CM comprises the amino acid sequence GPRSFGL (SEQ ID NO: 344). In some embodiments, the CM comprises the amino acid sequence GPRSFG (SEQ ID NO: 345).

In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of NTLSGRSENHSG (SEQ ID NO: 298); NTLSGRSGNHGS (SEQ ID NO: 299); TSTSGRSANPRG (SEQ ID NO: 300); TSGRSANP (SEQ ID NO: 301); VAGRSMRP (SEQ ID NO: 310); VVPEGRRS (SEQ ID NO: 311); ILPRSPAF (SEQ ID NO: 312); MVLGRSLL (SEQ ID NO: 313); QGRAITFI (SEQ ID NO: 314); SPRSIMLA (SEQ ID NO: 315); and SMLRSMPL (SEQ ID NO: 316).

In some embodiments, the CM comprises the amino acid sequence NTLSGRSENHSG (SEQ ID NO: 298). In some embodiments, the CM comprises the amino acid sequence NTLSGRSGNHGS (SEQ ID NO: 299). In some embodiments, the CM comprises the amino acid sequence TSTSGRSANPRG (SEQ ID NO: 300). In some embodiments, the CM comprises the amino acid sequence TSGRSANP (SEQ ID NO: 301). In some embodiments, the CM comprises the amino acid sequence VAGRSMRP (SEQ ID NO: 310). In some embodiments, the CM comprises the amino acid sequence VVPEGRRS (SEQ ID NO: 311). In some embodiments, the CM comprises the amino acid sequence ILPRSPAF (SEQ ID NO: 312). In some embodiments, the CM comprises the amino acid sequence MVLGRSLL (SEQ ID NO: 313). In some embodiments, the CM comprises the amino acid sequence QGRAITFI (SEQ ID NO: 314). In some embodiments, the CM comprises the amino acid sequence SPRSIMLA (SEQ ID NO: 315). In some embodiments, the CM comprises the amino acid sequence SMLRSMPL (SEQ ID NO: 316).

In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294-361, 1092-1112, 1157, 1158, 1161, 1162, 1165, 1166, 1169, 1520, and 1695-1704. In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294, 300, 302, 303, 305, 308, 318, 347, 361, 1092-1102, 1111, and 1157.

In some embodiments, the CM is a substrate for a neutrophil elastase. In some embodiments, the CM is a substrate for a serine protease. In some embodiments, the CM is a substrate for uPA. In some embodiments, the CM is a substrate for legumain. In some embodiments, the CM is a substrate for matriptase. In some embodiments, the CM is a substrate for a cysteine protease. In some embodiments, the CM is a substrate for a cysteine protease, such as a cathepsin.

In some embodiments, the CM is a CM1-CM2 substrate and includes the sequence ISSGLLSGRSDNH (SEQ ID NO: 214); ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 346); AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 347); TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 348); VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 349); TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 350); AVGLLAPPGGLSGRSDNH (SEQ ID NO: 318); LSGRSDNHGGAVGLLAPP (SEQ ID NO: 351); VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 352); LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 353); LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 354); LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 355); ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 356); LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 357); QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 358); LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 359); QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 360); ISSGLLSGRSGNH (SEQ ID NO: 361); ISSGLLSGRSANPRG (SEQ ID NO: 1092); AVGLLAPPTSGRSANPRG (SEQ ID NO: 1093); AVGLLAPPSGRSANPRG (SEQ ID NO: 1094); ISSGLLSGRSDDH (SEQ ID NO: 1095); ISSGLLSGRSDIH (SEQ ID NO: 1096); ISSGLLSGRSDQH (SEQ ID NO: 1097); ISSGLLSGRSDTH (SEQ ID NO: 1098); ISSGLLSGRSDDH (SEQ ID NO: 1099); ISSGLLSGRSDNP (SEQ ID NO: 1100); ISSGLLSGRSANP (SEQ ID NO: 1101); ISSGLLSGRSANI (SEQ ID NO: 1102); AVGLLAPPGGLSGRSDDH (SEQ ID NO: 1103); AVGLLAPPGGLSGRSDIH (SEQ ID NO: 1104); AVGLLAPPGGLSGRSDQH (SEQ ID NO: 1105); AVGLLAPPGGLSGRSDTH (SEQ ID NO: 1106); AVGLLAPPGGLSGRSDDH (SEQ ID NO: 1107); AVGLLAPPGGLSGRSDNP (SEQ ID NO: 1108); AVGLLAPPGGLSGRSANP (SEQ ID NO: 1109); AVGLLAPPGGLSGRSANI (SEQ ID NO: 1110); ISSGLLSGRSDNI (SEQ ID NO: 1111); and/or AVGLLAPPGGLSGRSDNI (SEQ ID NO: 1112).

In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDNH (SEQ ID NO: 214), which is also referred to herein as substrate 2001. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 346), which is also referred to herein as substrate 1001/LP′/0001, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 1519). In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 347), which is also referred to herein as substrate 1004/LP′/0003, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 348), which is also referred to herein as substrate 0003/LP′/1004, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 349), which is also referred to herein as substrate 1003/LP′/0003, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 350), which is also referred to herein as substrate 0003/LP′/1003, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDNH (SEQ ID NO: 318), which is also referred to herein as substrate 1004/LP′/0001, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence LSGRSDNHGGAVGLLAPP (SEQ ID NO: 351), which is also referred to herein as substrate 0001/LP′/1004, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 352), which is also referred to herein as substrate 1003/LP′/0001, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 353), which is also referred to herein as substrate 0001/LP′/1003, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 354), which is also referred to herein as substrate 0001/LP′/1001, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 1519). In some embodiments, the CM1-CM2 substrate includes the sequence LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 355), which is also referred to herein as substrate 0002/LP′/1001, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 1519). In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 356), which is also referred to herein as substrate 1001/LP′/0002, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 1519). In some embodiments, the CM1-CM2 substrate includes the sequence LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 357), which is also referred to herein as substrate 0001/LP′/1002, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 1519). In some embodiments, the CM1-CM2 substrate includes the sequence QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 358), which is also referred to herein as substrate 1002/LP′/0001, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 1519). In some embodiments, the CM1-CM2 substrate includes the sequence LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 359), which is also referred to herein as substrate 0002/LP′/1002, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 1519). In some embodiments, the CM1-CM2 substrate includes the sequence QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 360), which is also referred to herein as substrate 1002/LP′/0002, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GGSGGS (SEQ ID NO: 1519). In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSGNH (SEQ ID NO: 361), which is also referred to herein as substrate 2002. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSANPRG (SEQ ID NO: 1092), which is also referred to herein as substrate 2003. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPTSGRSANPRG (SEQ ID NO: 1093), which is also referred to herein as substrate 2004. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPSGRSANPRG (SEQ ID NO: 1094), which is also referred to herein as substrate 2005. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDDH (SEQ ID NO: 1095), which is also referred to herein as substrate 2006. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDIH (SEQ ID NO: 1096), which is also referred to herein as substrate 2007. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDQH (SEQ ID NO: 1097), which is also referred to herein as substrate 2008. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDTH (SEQ ID NO: 1098), which is also referred to herein as substrate 2009. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDDH (SEQ ID NO: 1099), which is also referred to herein as substrate 2010. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDNP (SEQ ID NO: 1100), which is also referred to herein as substrate 2011. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSANP (SEQ ID NO: 1101), which is also referred to herein as substrate 2012. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSANI (SEQ ID NO: 1102), which is also referred to herein as substrate 2013. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDDH (SEQ ID NO: 1103), which is also referred to herein as substrate 3006. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDIH (SEQ ID NO: 1104), which is also referred to herein as substrate 3007. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDQH (SEQ ID NO: 1105), which is also referred to herein as substrate 3008. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDTH (SEQ ID NO: 1106), which is also referred to herein as substrate 3009. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDDH (SEQ ID NO: 1107), which is also referred to herein as substrate 3010. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSDNP (SEQ ID NO: 1108), which is also referred to herein as substrate 3011. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSANP (SEQ ID NO: 1109), which is also referred to herein as substrate 3012. In some embodiments, the CM1-CM2 substrate includes the sequence AVGLLAPPGGLSGRSANI (SEQ ID NO: 1110), which is also referred to herein as substrate 3013. In some embodiments, the CM1-CM2 substrate includes the sequence ISSGLLSGRSDNI (SEQ ID NO: 1111), which is also referred to herein as substrate 2014. In some embodiments, the CM1-CM2 substrate includes the sequence and/or AVGLLAPPGGLSGRSDNI (SEQ ID NO: 1112), which is also referred to herein as substrate 3014. In some embodiments, the CM1-CM2 substrate includes the sequence GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 1970), which is also referred to herein as substrate 0001/LP′/1004, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GG. In some embodiments, the CM1-CM2 substrate includes the sequence GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 1971), which is also referred to herein as substrate 0001/LP′/1003, where LP′ as used in this CM1-CM2 substrate is the amino acid sequence GG.

In some embodiments, the CM is a substrate for at least two proteases. In some embodiments, each protease is selected from the group consisting of those shown in Table 3. In some embodiments, the CM is a substrate for at least two proteases, wherein one of the proteases is selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain and matriptase and the other protease is selected from the group consisting of those shown in Table 3. In some embodiments, the CM is a substrate for at least two proteases selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain and matriptase.

In some embodiments, the activatable antibody includes at least a first CM and a second CM. In some embodiments, the first CM and the second CM are each polypeptides of no more than 15 amino acids long. In some embodiments, the first CM and the second CM in the activatable antibody in the uncleaved state have the structural arrangement from N-terminus to C-terminus as follows: MM-CM1-CM2-AB or AB-CM2-CM1-MM. In some embodiments, at least one of the first CM and the second CM is a polypeptide that functions as a substrate for a protease selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain, and matriptase. In some embodiments, the first CM is cleaved by a first cleaving agent selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain, and matriptase in a target tissue and the second CM is cleaved by a second cleaving agent in a target tissue. In some embodiments, the other protease is selected from the group consisting of those shown in Table 3. In some embodiments, the first cleaving agent and the second cleaving agent are the same protease selected from the group consisting of a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain, and matriptase, and the first CM and the second CM are different substrates for the enzyme. In some embodiments, the first cleaving agent and the second cleaving agent are the same protease selected from the group consisting of those shown in Table 3. In some embodiments, the first cleaving agent and the second cleaving agent are different proteases. In some embodiments, the first cleaving agent and the second cleaving agent are co-localized in the target tissue. In some embodiments, the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue.

In some embodiments, the activatable antibody is exposed to and cleaved by a protease such that, in the activated or cleaved state, the activated antibody includes a light chain amino acid sequence that includes at least a portion of LP2 and/or CM sequence after the protease has cleaved the CM.

In some embodiments, the activatable antibody also includes an agent conjugated to the AB. In some embodiments, the agent conjugated to the AB or the AB of an activatable antibody is a therapeutic agent. In some embodiments, the agent is conjugated to the AB via a cleavable linker. In some embodiments, the agent is conjugated to the AB via a linker that includes at least one CM1-CM2 substrate sequence. In some embodiments, the agent is conjugated to the AB via a noncleavable linker.

In some embodiments, the agent is an anti-inflammatory agent.

In some embodiments, the activatable antibody also includes a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

In some embodiments, the activatable antibody also includes a signal peptide. In some embodiments, the signal peptide is conjugated to the activatable antibody via a spacer. In some embodiments, the spacer is conjugated to the activatable antibody in the absence of a signal peptide. In some embodiments, the spacer is joined directly to the MM of the activatable antibody. In some embodiments, the spacer is joined directly to the MM of the activatable antibody in the structural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB. An example of a spacer joined directly to the N-terminus of MM of the activatable antibody is selected from the group consisting of QGQSGQG (SEQ ID NO: 362), QGQSGQ (SEQ ID NO: 913), QGQSG (SEQ ID NO: 914), QGQS (SEQ ID NO: 915), QGQ (SEQ ID NO: 916), QG (SEQ ID NO: 917), and Q. Additional examples of spacers include GQSGQG (SEQ ID NO: 2042), QSGQG (SEQ ID NO: 2043), SGQG (SEQ ID NO: 2044), GQG(SEQ ID NO: 2045), QG(SEQ ID NO: 2046), and G. In some embodiments, the spacer includes at least the amino acid sequence QGQSGQG (SEQ ID NO: 362). In some embodiments, the spacer includes at least the amino acid sequence QGQSGQ (SEQ ID NO: 913). In some embodiments, the spacer includes at least the amino acid sequence QGQSG (SEQ ID NO: 914). In some embodiments, the spacer includes at least the amino acid sequence QGQS (SEQ ID NO: 915), In some embodiments, the spacer includes at least the amino acid sequence QGQ (SEQ ID NO: 916). In some embodiments, the spacer includes at least the amino acid sequence QG (SEQ ID NO: 917). In some embodiments, the spacer includes at least the amino acid residue Q. In some embodiments, the spacer includes at least the amino acid sequence GQSGQG (SEQ ID NO: 2042). In some embodiments, the spacer includes at least the amino acid sequence QSGQG (SEQ ID NO: 2043). In some embodiments, the spacer includes at least the amino acid sequence SGQG (SEQ ID NO: 2044). In some embodiments, the spacer includes at least the amino acid sequence GQG(SEQ ID NO: 2045). In some embodiments, the spacer includes at least the amino acid sequence QG (SEQ ID NO: 2046). In some embodiments, the spacer includes at least the amino acid sequence G. In some embodiments, the activatable antibody does not include a spacer sequence.

In some embodiments, the AB of the activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the AB can be engineered to include one or more disulfide bonds.

In some embodiments, the serum half-life of the activatable antibody is longer than that of the corresponding antibody; e.g., the pK of the activatable antibody is longer than that of the corresponding antibody. In some embodiments, the serum half-life of the activatable antibody is similar to that of the corresponding antibody. In some embodiments, the serum half-life of the activatable antibody is at least 15 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 12 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 11 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 10 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 9 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 8 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 7 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 5 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 2 days when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 24 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 20 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 18 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 16 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 14 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 12 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 10 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 8 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 6 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 4 hours when administered to an organism. In some embodiments, the serum half-life of the activatable antibody is at least 3 hours when administered to an organism.

In some embodiments, the activatable anti-PD-1 antibody is monospecific.

In some embodiments, the activatable anti-PD-1 antibody is multispecific, e.g., by way of non-limiting example, bispecific or trifunctional. In some embodiments, the activatable anti-PD-1 antibody is formulated as part of a pro-Bispecific T Cell Engager (BITE) molecule, i.e., the BITE includes a masking moiety and a cleavable moiety. In some embodiments, the activatable anti-PD-1 antibody is formulated as part of a pro-Chimeric Antigen Receptor (CAR) modified T cell, modified NK cell, or other modified immune effector cell. In some embodiments, an activatable anti-PD-1 antibody is formulated as part of another engineered receptor on an immune effector cell; i.e., the pro-CAR or other pro-engineered receptor includes a masking moiety and a cleavable moiety.

In some embodiments, the activatable antibody or antigen-binding fragment thereof is incorporated in a multispecific activatable antibody or antigen-binding fragment thereof, where at least one arm of the multispecific activatable antibody specifically binds PD-1. In some embodiments, the activatable antibody or antigen-binding fragment thereof is incorporated in a bispecific activatable antibody or antigen-binding fragment thereof, where at least one arm of the bispecific activatable antibody specifically binds PD-1.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, comprises a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, comprises a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, a heavy chain amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 45 or SEQ ID NO: 47. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21 and a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 45 or SEQ ID NO: 47. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21 and a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 45. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a heavy chain that comprises or is derived from amino acid sequence SEQ ID NO: 21 and a light chain that comprises or is derived from amino acid sequence SEQ ID NO: 47.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes the combination of the complementarity determining region (CDR) sequences of the heavy chain amino acid sequence of SEQ ID NO: 21. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes the combination of the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 45 or of SEQ ID NO: 47. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 21, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 45 or 47. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 21, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 45. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 21, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 47.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody includes: (a) a variable heavy chain complementarity determining region 1 (VH CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 653-657; (b) a variable heavy chain complementarity determining region 2 (VH CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 658-663; (c) a variable heavy chain complementarity determining region 3 (VH CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 664-668; (d) a variable light chain complementarity determining region 1 (VL CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 669-677; (e) a variable light chain complementarity determining region 2 (VL CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 678-682; and (f) variable light chain complementarity determining region 3 (VL CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 683-687

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664).

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675) or RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VL CDR1 sequence comprises RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683).

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664); the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675) or RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664); the VL CDR1 sequence comprises RASESVDSYGISFMN (SEQ ID NO: 675); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683). In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GFTFSGYAMS (SEQ ID NO: 653); the VH CDR2 sequence comprises YISNSGGNAH (SEQ ID NO: 658); and the VH CDR3 sequence comprises EDYGTSPFVY (SEQ ID NO: 664); the VL CDR1 sequence comprises RASESVDAYGISFMN (SEQ ID NO:676); the VL CDR2 sequence comprises AASNQGS (SEQ ID NO: 678); and the VL CDR3 sequence comprises QQSKDVPWT (SEQ ID NO: 683).

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a heavy chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 1346, and a light chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 626.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a heavy chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 1514, and a light chain that comprises or is derived from the amino acid sequence of SEQ ID NO: 638.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes the combination of the complementarity determining region (CDR) sequences of the heavy chain amino acid sequence of SEQ ID NO: 1346. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes the combination of the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 626. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 1346, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 626.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, wherein the VH CDR1 sequence comprises GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises IWYDGSKR (SEQ ID NO: 1706); and the VH CDR3 sequence comprises TNDDY (SEQ ID NO: 1707).

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein the VL CDR1 sequence comprises QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises QQSSNWPRT (SEQ ID NO: 1710).

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GITFSNSG (SEQ ID NO: 1705); the VH CDR2 sequence comprises IWYDGSKR (SEQ ID NO: 1706); the VH CDR3 sequence comprises TNDDY (SEQ ID NO: 1707); the VL CDR1 sequence comprises QSVSSY (SEQ ID NO: 1708); the VL CDR2 sequence comprises DAS (SEQ ID NO: 1709); and the VL CDR3 sequence comprises QQSSNWPRT (SEQ ID NO: 1710).

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes the combination of the complementarity determining region (CDR) sequences of the heavy chain amino acid sequence of SEQ ID NO: 1514. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes the combination of the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 638. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of the CDR sequences of the heavy chain amino acid sequence of SEQ ID NO: 1514, and the CDR sequences of the light chain amino acid sequence of SEQ ID NO: 638.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, and a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, wherein the VH CDR1 sequence comprises GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises INPSNGGT (SEQ ID NO: 1712); and the VH CDR3 sequence comprises RRDYRFDMGFDY (SEQ ID NO: 1713).

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein the VL CDR1 sequence comprises KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises QHSRDLPLT (SEQ ID NO: 1716).

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprises GYTFTNYY (SEQ ID NO: 1711); the VH CDR2 sequence comprises INPSNGGT (SEQ ID NO: 1712); the VH CDR3 sequence comprises RRDYRFDMGFDY (SEQ ID NO: 1713); the VL CDR1 sequence comprises KGVSTSGYSY (SEQ ID NO: 1714); the VL CDR2 sequence comprises LAS (SEQ ID NO: 1715); and the VL CDR3 sequence comprises QHSRDLPLT (SEQ ID NO: 1716).

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, comprises a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, comprises a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1346, and a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 626.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, includes a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1514, and a light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 638.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, comprises a combination of a variable heavy chain complementarity determining region 1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavy chain complementarity determining region 2 (VH CDR2, also referred to herein as CDRH2) sequence, a variable heavy chain complementarity determining region 3 (VH CDR3, also referred to herein as CDRH3) sequence, a variable light chain complementarity determining region 1 (VL CDR1, also referred to herein as CDRL1) sequence, a variable light chain complementarity determining region 2 (VL CDR2, also referred to herein as CDRL2) sequence, and a variable light chain complementarity determining region 3 (VL CDR3, also referred to herein as CDRL3) sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence shown in Table 8; a VH CDR2 sequence shown in Table 8; a VH CDR3 sequence shown in Table 8; a VL CDR1 sequence shown in Table 8; a VL CDR2 sequence shown in Table 8; and a VL CDR3 sequence shown in Table 8.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one CDR sequence is selected from the group consisting of a VH CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR1 sequence shown in Table 8; a VH CD2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR2 sequence shown in Table 8; a VH CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VH CDR3 sequence shown in Table 8; a VL CDR1 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR1 sequence shown in Table 8; a VL CDR2 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR2 sequence shown in Table 8; and a VL CDR3 sequence that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a VL CDR3 sequence shown in Table 8.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence shown in a single row in Table 8, and a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence shown in a single row in Table 8.

In some embodiments, the antibody comprises a heavy chain that comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the combination is a combination of the three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single row in Table 8.

In some embodiments, at least one arm of the multispecific activatable antibody, e.g., a bispecific activatable antibody, comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence shown in a single row in Table 8, and a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence shown in a single row in Table 8, wherein each CDR sequence in the combination comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the corresponding VH CDR sequence shown in a single row in Table 8 and the corresponding VL CDR sequence shown in a single row in Table 8.

In some embodiments, the antibody comprises a heavy chain that comprises a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein each CDR sequence in the combination comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the corresponding CDR sequence in a combination of three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single row in Table 8.

In some embodiments, the anti-PD-1 antibodies and/or activatable anti-PD-1 antibodies described herein are used in conjunction with one or more additional agents or a combination of additional agents. Suitable additional agents include other experimental anti-cancer agents in development for specific applications, current pharmaceutical and/or surgical therapies for an intended application, such as, for example, cancer. For example, the anti-PD-1 antibodies and/or activatable anti-PD-1 antibodies can be used in conjunction with an additional chemotherapeutic or anti-neoplastic agent.

In some embodiments, the additional agent includes at least one experimental anti-cancer agent in development for specific applications. In some embodiments, the additional agent includes at least one agent that is already approved for one indication but is being tested in another. In some embodiments, the additional agent includes at least one agent that is not currently approved in any indication but is being tested in one or more indications for the purposes of obtaining regulatory approval. In some embodiments, the additional agent includes at least one agent that is a pharmaceutical therapy for an intended application. In some embodiments, the additional agent includes at least one agent that is a surgical therapy for cancer. In some embodiments, the additional agent includes at least one agent that is a pharmaceutical therapy for an intended application. In some embodiments, the additional agent includes at least one agent that is a surgical therapy for cancer. In some embodiments, the additional agent is a chemotherapeutic agent. In some embodiments, the additional agent is an anti-neoplastic agent. In some embodiments, the additional agents are a combination of any two or more of these agents.

In some embodiments, the additional agent(s) is a chemotherapeutic agent, such as a chemotherapeutic agent selected from the group consisting of docetaxel, paclitaxel, abraxane (i.e., albumin-conjugated paclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin, irinotecan, and gemcitabine.

In some embodiments, the additional agent(s) is a checkpoint inhibitor, a kinase inhibitor, an agent targeting inhibitors in the tumor microenvironment, and/or a T cell or NK agonist. In some embodiments, the additional agent(s) is radiation therapy, alone or in combination with another additional agent(s) such as a chemotherapeutic or anti-neoplastic agent. In some embodiments, the additional agent(s) is a vaccine, an oncovirus, and/or a DC-activating agent such as, by way of non-limiting example, a toll-like receptor (TLR) agonist and/or α-CD40. In some embodiments, the additional agent(s) is a tumor-targeted antibody designed to kill the tumor via ADCC or via direct conjugation to a toxin (e.g., an antibody drug conjugate (ADC).

In some embodiments, the checkpoint inhibitor is an inhibitor of a target selected from the group consisting of CTLA-4, LAG-3, PD-1, PD-1, TIGIT, TIM-3, B7H4, BTLA, and Vista. In some embodiments, the kinase inhibitor is selected from the group consisting of B-RAFi, MEKi, and Btk inhibitors, such as ibrutinib. In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the B-RAF inhibitor is vemurafenib. In some embodiments, the tumor microenvironment inhibitor is selected from the group consisting of an IDO inhibitor, an α-CSF1R inhibitor, an α-CCR4 inhibitor, a TGF-beta, a myeloid-derived suppressor cell, or a T-regulatory cell. In some embodiments, the agonist is selected from the group consisting of Ox40, GITR, CD137, ICOS, CD27, and HVEM.

In some embodiments, the inhibitor is a CTLA-4 inhibitor. In some embodiments, the inhibitor is a LAG-3 inhibitor. In some embodiments, the inhibitor is a PD-1 inhibitor. In some embodiments, the inhibitor is a PD-1 inhibitor. In some embodiments, the inhibitor is a TIGIT inhibitor. In some embodiments, the inhibitor is a TIM-3 inhibitor. In some embodiments, the inhibitor is a B7H4 inhibitor. In some embodiments, the inhibitor is a Vista inhibitor. In some embodiments, the inhibitor is a B-RAFi inhibitor. In some embodiments, the inhibitor is a MEKi inhibitor. In some embodiments, the inhibitor is a Btk inhibitor. In some embodiments, the inhibitor is ibrutinib. In some embodiments, the inhibitor is crizotinib. In some embodiments, the inhibitor is an IDO inhibitor. In some embodiments, the inhibitor is an α-CSF1R inhibitor. In some embodiments, the inhibitor is an α-CCR4 inhibitor. In some embodiments, the inhibitor is a TGF-beta. In some embodiments, the inhibitor is a myeloid-derived suppressor cell. In some embodiments, the inhibitor is a T-regulatory cell.

In some embodiments, the agonist is O×40. In some embodiments, the agonist is GITR. In some embodiments, the agonist is CD137. In some embodiments, the agonist is ICOS. In some embodiments, the agonist is CD27. In some embodiments, the agonist is HVEM.

In some embodiments, the anti-PD-1 antibody and/or activatable antibody is administered during and/or after treatment in combination with one or more additional agents such as, for example, a chemotherapeutic agent, an anti-inflammatory agent, and/or an immunosuppressive agent. In some embodiments, the anti-PD-1 antibody and/or activatable anti-PD-1 antibody and the additional agent are formulated into a single therapeutic composition, and the anti-PD-1 antibody and/or activatable anti-PD-1 antibody and additional agent are administered simultaneously. Alternatively, the anti-PD-1 antibody and/or activatable anti-PD-1 antibody and additional agent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and the anti-PD-1 antibody and/or activatable anti-PD-1 antibody and the additional agent are administered simultaneously, or the anti-PD-1 antibody and/or activatable anti-PD-1 antibody and the additional agent are administered at different times during a treatment regimen. For example, the anti-PD-1 antibody and/or activatable anti-PD-1 antibody is administered prior to the administration of the additional agent, the anti-PD-1 antibody and/or activatable anti-PD-1 antibody is administered subsequent to the administration of the additional agent, or the anti-PD-1 antibody and/or activatable anti-PD-1 antibody and the additional agent are administered in an alternating fashion. As described herein, the anti-PD-1 antibody and/or activatable anti-PD-1 antibody and additional agent are administered in single doses or in multiple doses.

In some embodiments, the anti-PD-1 antibody and/or activatable anti-PD-1 antibody and the additional agent(s) are administered simultaneously. For example, the anti-PD-1 antibody and/or activatable anti-PD-1 antibody and the additional agent(s) can be formulated in a single composition or administered as two or more separate compositions. In some embodiments, the anti-PD-1 antibody and/or activatable anti-PD-1 antibody and the additional agent(s) are administered sequentially, or the anti-PD-1 antibody and/or activatable anti-PD-1 antibody and the additional agent are administered at different times during a treatment regimen.

In some embodiments, the anti-PD-1 antibody and/or activatable anti-PD-1 antibody is administered during and/or after treatment in combination with one or more additional agents such as, by way of non-limiting example, a chemotherapeutic agent, an anti-inflammatory agent, and/or an immunosuppressive agent, such as an alkylating agent, an anti-metabolite, an anti-microtubule agent, a topoisomerase inhibitor, a cytotoxic antibiotic, and/or any other nucleic acid damaging agent. In some embodiments, the additional agent is a taxane, such as paclitaxel (e.g., Abraxane®). In some embodiments, the additional agent is an anti-metabolite, such as gemcitabine. In some embodiments, the additional agent is an alkylating agent, such as platinum-based chemotherapy, such as carboplatin or cisplatin. In some embodiments, the additional agent is a targeted agent, such as a kinase inhibitor, e.g., sorafenib or erlotinib. In some embodiments, the additional agent is a targeted agent, such as another antibody, e.g., a monoclonal antibody (e.g., bevacizumab), a bispecific antibody, or a multispecific antibody. In some embodiments, the additional agent is a proteosome inhibitor, such as bortezomib or carfilzomib. In some embodiments, the additional agent is an immune modulating agent, such as lenolidominde or IL-2. In some embodiments, the additional agent is radiation. In some embodiments, the additional agent is an agent considered standard of care by those skilled in the art. In some embodiments, the additional agent is a chemotherapeutic agent well known to those skilled in the art.

In some embodiments, the additional agent is another antibody or antigen-binding fragment thereof and/or another activatable antibody or antigen-binding fragment thereof. In some embodiments the additional agent is another antibody or antigen-binding fragment thereof and/or another activatable antibody or antigen-binding fragment thereof against the same target as the first antibody or antigen-binding fragment thereof and/or an activatable antibody or antigen-binding fragment thereof, e.g., against PD-1. In some embodiments the additional agent is another antibody or antigen-binding fragment thereof and/or another activatable antibody or antigen-binding fragment thereof against a target different than the target of the first antibody or antigen-binding fragment thereof, the first conjugated antibody or antigen-binding fragment thereof and/or activatable antibody or antigen-binding fragment thereof.

As a non-limiting example, the antibody or antigen-binding fragment and/or the AB of an activatable antibody is a binding partner for any target listed in Table 1.

TABLE 1 Exemplary Targets 1-92-LFA-3 CD52 DL44 HVEM LIF-R STEAP1 Alpha-4 CD56 DLK1 Hyaluronidase Lewis X STEAP2 integrin Alpha-V CD64 DLL4 ICOS LIGHT TAG-72 integrin alpha4beta1 CD70 DPP-4 IFNalpha LRP4 TAPA1 integrin alpha4beta7 CD71 DSG1 IFNbeta LRRC26 TGFbeta integrin AGR2 CD74 EGFR IFNgamma MCSP TIGIT Anti-Lewis-Y EGFRviii IgE Mesothelin TIM-3 Apelin J CD80 Endothelin B IgE Receptor MRP4 TLR2 receptor receptor (FceRI) (ETBR) APRIL CD81 ENPP3 IGF MUC1 TLR4 B7-H4 CD86 EpCAM IGF1R Mucin-16 TLR6 (MUC16, CA-125) BAFF CD95 EPHA2 IL1B Na/K ATPase TLR7 BTLA CD117 EPHB2 IL1R Neutrophil TLR8 elastase C5 CD125 ERBB3 IL2 NGF TLR9 complement C-242 CD132 F protein of IL11 Nicastrin TMEM31 (IL-2RG) RSV CA9 CD133 FAP IL12 Notch TNFalpha Receptors CA19-9 CD137 FGF-2 IL12p40 Notch 1 TNFR (Lewis a) Carbonic CD138 FGF8 IL-12R, Notch 2 TNFRS12 anhydrase 9 IL-12Rbeta1 A CD2 CD166 FGFR1 IL13 Notch 3 TRAIL-R1 CD3 CD172A FGFR2 IL13R Notch 4 TRAIL-R2 CD6 CD248 FGFR3 IL15 NOV Transferrin CD9 CDH6 FGFR4 IL17 OSM-R Transferrin receptor CD11a CEACAM5 Folate IL18 OX-40 TRK-A (CEA) receptor CD19 CEACAM6 GAL3ST1 IL21 PAR2 TRK-B (NCA-90) CD20 CLAUDIN-3 G-CSF IL23 PDGF-AA uPAR CD22 CLAUDIN-4 G-CSFR IL23R PDGF-BB VAP1 CD24 cMet GD2 IL27/IL27R PDGFRalpha VCAM-1 (wsx1) CD25 Collagen GITR IL29 PDGFRbeta VEGF CD27 Cripto GLUT1 IL-31R PD-1 VEGF-A CD28 CSFR GLUT4 IL31/IL31R PD-L1 VEGF-B CD30 CSFR-1 GM-CSF IL2R PD-L2 VEGF-C CD33 CTLA-4 GM-CSFR IL4 Phosphatidyl- VEGF-D serine CD38 CTGF GP IIb/IIIa IL4R P1GF VEGFR1 receptors CD40 CXCL10 Gp130 IL6, IL6R PSCA VEGFR2 CD40L CXCL13 GPIIB/IIIA Insulin PSMA VEGFR3 Receptor CD41 CXCR1 GPNMB Jagged RAAG12 VISTA Ligands CD44 CXCR2 GRP78 Jagged 1 RAGE WISP-1 CD44v6 HER2/neu Jagged 2 SLC44A4 WISP-2 CD47 CXCR4 HGF LAG-3 Sphingosine 1 WISP-3 Phosphate CD51 CYR61 hGH

As a non-limiting example, the antibody or antigen-binding fragment and/or the AB of an activatable antibody is or is derived from an antibody listed in Table 2.

TABLE 2 Exemplary sources for Abs Antibody Trade Name (antibody name) Target Avastin ™ (bevacizumab) VEGF Lucentis ™ (ranibizumab) VEGF Erbitux ™ (cetuximab) EGFR Vectibix ™ (panitumumab) EGFR Remicade ™ (infliximab) TNFα Humira ™ (adalimumab) TNFα Tysabri ™ (natalizumab) Integrinα4 Simulect ™ (basiliximab) IL2R Soliris ™ (eculizumab) Complement C5 Raptiva ™ (efalizumab) CD11a Bexxar ™ (tositumomab) CD20 Zevalin ™ (ibritumomab tiuxetan) CD20 Rituxan ™ (rituximab) CD20 Ocrelizumab CD20 Arzerra ™ (ofatumumab) CD20 Gazyva ™ (Obinutuzumab) CD20 Zenapax ™ (daclizumab) CD25 Adcetris ™ (brentuximab vedotin) CD30 Myelotarg ™ (gemtuzumab) CD33 Mylotarg ™ (gemtuzumab ozogamicin) CD33 Campath ™ (alemtuzumab) CD52 ReoPro ™ (abiciximab) Glycoprotein receptor IIb/IIIa Xolair ™ (omalizumab) IgE Herceptin ™ (trastuzumab) Her2 Kadcyla ™ (trastuzumab emtansine) Her2 Synagis ™ (palivizumab) F protein of RSV (ipilimumab) CTLA-4 (tremelimumab) CTLA-4 Hu5c8 CD40L (pertuzumab) Her2-neu (ertumaxomab) CD3/Her2-neu Orencia ™ (abatacept) CTLA-4 (tanezumab) NGF (bavituximab) Phosphatidylserine (zalutumumab) EGFR (mapatumumab) EGFR (matuzumab) EGFR (nimotuzumab) EGFR ICR62 EGFR mAb 528 EGFR CH806 EGFR MDX-447 EGFR/CD64 (edrecolomab) EpCAM RAV12 RAAG12 huJ591 PSMA Enbrel ™ (etanercept) TNF-R Amevive ™ (alefacept) 1-92-LFA-3 Antril ™, Kineret ™ (ankinra) IL-1Ra GC1008 TGFbeta Notch, e.g., Notch 1 Jagged 1 or Jagged 2 (adecatumumab) EpCAM (figitumumab) IGF1R (tocilizumab) IL-6 receptor Stelara ™ (ustekinumab) IL-12/IL-23 Prolia ™ (denosumab) RANKL

In some embodiments, the additional antibody or antigen binding fragment thereof and/or activatable antibody or antigen binding fragment thereof is a monoclonal antibody, domain antibody, single chain, Fab fragment, a F(ab′)₂ fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody. In some embodiments, the additional antibody or antigen binding fragment thereof and/or activatable antibody or antigen binding fragment thereof is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody.

The disclosure also provides methods of producing an anti-PD-1 antibody and/or activatable anti-PD-1 antibody polypeptide by culturing a cell under conditions that lead to expression of the polypeptide, wherein the cell comprises an isolated nucleic acid molecule encoding an antibody and/or an activatable antibody described herein, and/or vectors that include these isolated nucleic acid sequences. The disclosure provides methods of producing an antibody and/or activatable antibody by culturing a cell under conditions that lead to expression of the antibody and/or activatable antibody, wherein the cell comprises an isolated nucleic acid molecule encoding an antibody and/or an activatable antibody described herein, and/or vectors that include these isolated nucleic acid sequences.

The invention also provides a method of manufacturing activatable antibodies that in an activated state binds PD-1 by (a) culturing a cell comprising a nucleic acid construct that encodes the activatable antibody under conditions that lead to expression of the activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM), and an antibody or an antigen binding fragment thereof (AB) that specifically binds PD-1, (i) wherein the CM is a polypeptide that functions as a substrate for a protease; and (ii) wherein the CM is positioned in the activatable antibody such that, when the activatable antibody is in an uncleaved state, the MM interferes with specific binding of the AB to PD-1 and in a cleaved state the MM does not interfere or compete with specific binding of the AB to PD-1; and (b) recovering the activatable antibody. Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein.

In some embodiments, the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM. In some embodiments, the activatable antibody comprises a linking peptide between the MM and the CM. In some embodiments, the activatable antibody comprises a linking peptide between the CM and the AB. In some embodiments, the activatable antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: spacer-MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM-spacer.

In some embodiments, the two linking peptides need not be identical to each other.

In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 363) and (GGGS)_(n) (SEQ ID NO: 364), where n is an integer of at least one.

In some embodiments, at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 365), GGSGG (SEQ ID NO: 366), GSGSG (SEQ ID NO: 367), GSGGG (SEQ ID NO: 368), GGGSG (SEQ ID NO: 369), and GSSSG (SEQ ID NO: 370).

In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 371), GSSGGSGGSGG (SEQ ID NO: 372), GSSGGSGGSGGS (SEQ ID NO: 373), GSSGGSGGSGGSGGGS (SEQ ID NO: 374), GSSGGSGGSG (SEQ ID NO: 375), GSSGGSGGSGS (SEQ ID NO: 376), GGGSSGGS (SEQ ID NO: 65), or GGGSSGG (SEQ ID NO: 1040).

In some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 377), GSSGT (SEQ ID NO: 378) or GSSG (SEQ ID NO: 379).

The invention provides methods of preventing, delaying the progression of, treating, alleviating a symptom of, or otherwise ameliorating a disorder or disease in a subject by administering to a subject in need thereof a therapeutically effective amount of an anti-PD-1 antibody, conjugated anti-PD-1 antibody, activatable anti-PD-1 antibody and/or conjugated activatable anti-PD-1 antibody described herein.

The invention also provides a method of reducing immune suppression comprising administering to a subject in need thereof a therapeutically effective amount of an anti-PD-1 antibody and/or activatable anti-PD-1 antibody described herein. In some embodiments, the immune suppression is suppression of T cell activity. In some embodiments, the immune suppression is mediated by engagement of PD-1 on T cells to PD-L1 or PD-L2 on tumor cells or other immune cells. In some embodiments, the invention provides a method to reduce or inhibit binding of PD-L1 (also referred to herein as PDL1) and/or PD-L2 (also referred to herein as PDL2), to PD-1 on T cells. The ligands PD-L1 and/or PD-L2 are often found on tumor cells or other immune cells.

The invention also provides methods of preventing, delaying the progression of, treating, alleviating a symptom of, or otherwise ameliorating cancer in a subject by administering a therapeutically effective amount of an anti-PD-1 antibody and/or activatable anti-PD-1 antibody described herein to a subject in need thereof. PD-1 is known to be expressed on immune cells, such as T cells, in a variety of cancers, such as, by way of non-limiting example, melanoma, non-small cell lung cancer, nasopharyngeal cancer, glioblastoma/mixed glioma, colon adenocarcinoma, hepatocellular carcinoma, urothelial cancer, multiple myeloma, ovarian cancer, gastric carcinoma, esophageal cancer, pancreatic cancer, renal cell carcinoma (RCC), breast cancer, lymphomas, such as Hodgkin's lymphoma, and leukemias. (See e.g., Chen et al., “Molecular Pathways: Next-Generation Immunotherapy—Inhibiting Programmed Death-Ligand 1 and Programmed Death-1,” Clin. Can. Res., vol. 18: 6580-6587 (2012), the contents of which are hereby incorporated by reference in their entirety).

In some embodiments, the cancer is a bladder cancer, a bone cancer, a breast cancer, a carcinoid, a cervical cancer, a cholangiocarcinoma, a colon cancer, an endometrial cancer, a glioma, a head and neck cancer, a liver cancer, a lung cancer, a lymphoma, such as Hodgkin's lymphoma, a melanoma, an ovarian cancer, a pancreatic cancer, a prostate cancer, a renal cancer, a sarcoma, a skin cancer, a stomach cancer, a testis cancer, a thymus cancer, a thyroid cancer, a urogenital cancer, and/or a urothelial cancer.

In some embodiments, the cancer is selected from the group consisting of melanoma (MEL), renal cell carcinoma (RCC), squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer (CRC), castration-resistant prostate cancer (CRPC), hepatocellular carcinoma (HCC), squamous cell carcinoma of the head and neck, thymoma, carcinomas of the esophagus, ovary, gastrointestinal tract and breast, or a hematologic malignancy such as multiple myeloma, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, primary mediastinal B-cell lymphoma, and chronic myelogenous leukemia.

The invention also provides methods of treating cancer patients with an autoimmune or inflammatory disease by administering a therapeutically effective amount of an anti-PD-1 antibody and/or activatable anti-PD-1 antibody described herein to a subject in need thereof. In some embodiments, the autoimmune disease is colitis, RA, pancreatitis, diabetes, or pneumonitis.

An anti-PD-1 antibody and/or an activatable anti-PD-1 antibody used in any of the embodiments of these methods and uses can be administered at any stage of the disease. For example, such an anti-PD-1 antibody and/or activatable anti-PD-1 antibody can be administered to a patient suffering cancer of any stage, from early to metastatic. The terms subject and patient are used interchangeably herein.

In some embodiments, the subject is a mammal, such as a human, non-human primate, companion animal (e.g., cat, dog, horse), farm animal, work animal, or zoo animal. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal. In some embodiments, the subject is an animal in the care of a veterinarian.

The anti-PD-1 antibody and/or activatable anti-PD-1 antibody and therapeutic formulations thereof are administered to a subject suffering from or susceptible to a disease or disorder associated with immune suppression, such as immune suppression mediated by engagement of PD-1 on T cells by PD-L1 or PD-L2 on tumor cells or other immune cells. In some embodiments, the immune suppression is suppression of T cell activity. A subject suffering from or susceptible to a disease or disorder associated with such immune suppression is identified using any of a variety of methods known in the art. For example, subjects suffering from cancer or other neoplastic condition are identified using any of a variety of clinical and/or laboratory tests such as, physical examination and blood, urine and/or stool analysis to evaluate health status. For example, subjects suffering from inflammation and/or an inflammatory disorder are identified using any of a variety of clinical and/or laboratory tests such as physical examination and/or bodily fluid analysis, e.g., blood, urine and/or stool analysis, to evaluate health status.

Administration of an anti-PD-1 antibody and/or activatable anti-PD-1 antibody to a patient suffering from a disease or disorder associated with immune suppression, such as immune suppression mediated by engagement of PD-1 on T cells by PD-L1 or PD-L2 on tumor cells or other immune cells, is considered successful if any of a variety of laboratory or clinical objectives is achieved. For example, administration of an anti-PD-1 antibody, and/or activatable anti-PD-1 antibody to a patient suffering from a disease or disorder associated with such immune suppression is considered successful if one or more of the symptoms associated with the disease or disorder is alleviated, reduced, inhibited or does not progress to a further, i.e., worse, state. Administration of an anti-PD-1 antibody and/or conjugated activatable anti-PD-1 antibody to a patient suffering from a disease or disorder associated with immune suppression, such as immune suppression mediated by engagement of PD-1 on T cells to PD-L1 or PD-L2 on tumor cells or other immune cells, is considered successful if the disease or disorder enters remission or does not progress to a further, i.e., worse, state.

In some embodiments, the anti-PD-1 antibody and/or conjugated activatable anti-PD-1 antibody is administered during and/or after treatment in combination with one or more additional agents such as, for example, a chemotherapeutic agent, an anti-inflammatory agent, and/or an immunosuppressive agent. In some embodiments, the anti-PD-1 antibody and/or activatable anti-PD-1 antibody and the additional agent(s) are administered simultaneously. For example, the anti-PD-1 antibody and/or activatable anti-PD-1 antibody and the additional agent(s) can be formulated in a single composition or administered as two or more separate compositions. In some embodiments, the anti-PD-1 antibody and/or activatable anti-PD-1 antibody and the additional agent(s) are administered sequentially.

The invention also provides methods and kits for using the activatable anti-PD-1 antibodies in a variety of diagnostic and/or prophylactic indications. For example, the invention provides methods and kits for detecting the presence or absence of a cleaving agent and a target of interest in a subject or a sample by (i) contacting a subject or sample with an anti-PD-1 activatable antibody, wherein the anti-PD-1 activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, and an antigen binding domain or fragment thereof (AB) that specifically binds the target of interest, wherein the anti-PD-1 activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to PD-1, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form of a natural binding partner of the AB; and (b) wherein, when the AB is in an uncleaved, non-activated state, the MM interferes with specific binding of the AB to PD-1, and when the AB is in a cleaved, activated state the MM does not interfere or compete with specific binding of the AB to PD-1; and (ii) measuring a level of activated anti-PD-1 activatable antibody in the subject or sample, wherein a detectable level of activated anti-PD-1 activatable antibody in the subject or sample indicates that the cleaving agent and PD-1 are present in the subject or sample and wherein no detectable level of activated anti-PD-1 activatable antibody in the subject or sample indicates that the cleaving agent, PD-1 or both the cleaving agent and PD-1 are absent in the subject or sample.

In some embodiments, the activatable anti-PD-1 antibody is an activatable anti-PD-1 antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable anti-PD-1 antibody is not conjugated to an agent. In some embodiments, the activatable anti-PD-1 antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable anti-PD-1 antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.

In some embodiments of these methods and kits, the activatable anti-PD-1 antibody includes a detectable label. In some embodiments of these methods and kits, the detectable label includes an imaging agent, a contrasting agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments of these methods and kits, the imaging agent comprises a radioisotope. In some embodiments of these methods and kits, the radioisotope is indium or technetium. In some embodiments of these methods and kits, the contrasting agent comprises iodine, gadolinium or iron oxide. In some embodiments of these methods and kits, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments of these methods and kits, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In some embodiments of these methods and kits, the luminescent label comprises an N-methylacrydium derivative. In some embodiments of these methods, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments of these methods and kits, the ligand-based label comprises biotin, avidin, streptavidin or one or more haptens.

In some embodiments of these methods and kits, the subject is a mammal. In some embodiments of these methods, the subject is a human. In some embodiments, the subject is a non-human mammal, such as a non-human primate, companion animal (e.g., cat, dog, horse), farm animal, work animal, or zoo animal. In some embodiments, the subject is a rodent.

In some embodiments of these methods and kits, the method is an in vivo method. In some embodiments of these methods, the method is an in situ method. In some embodiments of these methods, the method is an ex vivo method. In some embodiments of these methods, the method is an in vitro method.

In some embodiments of the methods and kits, the method is used to identify or otherwise refine a patient population suitable for treatment with an anti-PD-1 activatable antibody of the disclosure, followed by treatment by administering that activatable anti-PD-1 antibody to a subject in need thereof. For example, patients that test positive for both the target (e.g., PD-1) and a protease that cleaves the substrate in the cleavable moiety (CM) of the anti-PD-1 activatable antibody being tested in these methods are identified as suitable candidates for treatment with such an anti-PD-1 activatable antibody comprising such a CM, and the patient is then administered a therapeutically effective amount of the activatable anti-PD-1 antibody that was tested. Likewise, patients that test negative for either or both of the target (e.g., PD-1) and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods might be identified as suitable candidates for another form of therapy. In some embodiments, such patients can be tested with other anti-PD-1 activatable antibodies until a suitable anti-PD-1 activatable antibody for treatment is identified (e.g., an anti-PD-1 activatable antibody comprising a CM that is cleaved by the patient at the site of disease). In some embodiments, the patient is then administered a therapeutically effective amount of the activatable anti-PD-1 antibody for which the patient tested positive. Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein.

Pharmaceutical compositions according to the invention can include an antibody of the invention and a carrier. These pharmaceutical compositions can be included in kits, such as, for example, diagnostic kits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is graph depicting the results of an ELISA binding assay using various murine antibodies that bind human PD-1 (i.e., anti-hPD-1 antibodies) referred to herein as 245-M3, also referred to herein as M3 (VH: SEQ ID NO: 9; VL: SEQ ID NO: 11), 245-M5, also referred to herein as M5 (VH: SEQ ID NO: 13; VL: SEQ ID NO: 15), 136-M13, also referred to herein as M13 (VH: SEQ ID NO: 1; VL: SEQ ID NO: 3), 136-M19, also referred to herein as M19 (VH: SEQ ID NO: 5; VL: SEQ ID NO: 7), and 136-M14, also referred to herein as M14 (VH: SEQ ID NO: 17; VL: SEQ ID NO: 19). The anti-PD-1 antibodies nivolumab (also referred to herein as “nivo” or “NV1”) and pembrolizumab (also referred to herein as “pembro” or “PM1” or “PM1 AB”) were used as positive controls.

FIG. 2 is a series of graphs demonstrating that various murine anti-hPD-1 antibodies block binding of the ligands human PD-L1 (hPD-L1) and human PD-L2 (hPD-L2) to human PD-1.

FIG. 3 is a series of graphs demonstrating that various murine anti-hPD-1 antibodies block binding of the ligands human PD-L1 (hPD-L1) and human PD-L2 (hPD-L2) to human PD-1.

FIG. 4 is a graph depicting binding to human PD-1 of various anti-hPD-1 antibodies of the disclosure referred to herein as A1.0 (VH: SEQ ID NO: 21; VL: SEQ ID NO: 39), A1.2 (VH: SEQ ID NO: 21; VL: SEQ ID NO: 43), and A1.4 (VH: SEQ ID NO: 21; VL: SEQ ID NO: 45) as determined by ELISA. The anti-PD-1 antibody pembrolizumab (“PM1”) was used as positive control.

FIG. 5 is a graph depicting binding to human PD-1 of various anti-hPD-1 antibodies of the disclosure referred to herein as A1.5 (VH: SEQ ID NO: 21; VL: SEQ ID NO: 47), A1.6 (VH: SEQ ID NO: 21; VL: SEQ ID NO: 49), Ba2 (VH: SEQ ID NO: 29; VL: SEQ ID NO: 57), Bb2 (VH: SEQ ID NO: 31; VL: SEQ ID NO: 57), C1.1 (VH: SEQ ID NO: 33; VL: SEQ ID NO: 41), and D4 (VH: SEQ ID NO: 37; VL: SEQ ID NO: 59) as determined by ELISA. The anti-PD-1 antibodies nivolumab (“NV1”) and pembrolizumab (“PM1”) were used as positive controls.

FIG. 6 is a series of graphs depicting the ability of various anti-hPD-1 antibodies of the disclosure referred to herein as A1.0, A1.2, and A1.4 to inhibit binding between human PD-1 (hPD-1) and both human PDL-1 (hPDL-1) and human PDL-2 (hPDL-2) as determined by ELISA. The anti-PD-1 antibody pembrolizumab (“PM1”) was used as positive control.

FIG. 7 is a series of graphs depicting the ability of various anti-hPD-1 antibodies of the disclosure referred to herein as A1.5 and C1.1 to inhibit binding between hPD-1 and both hPDL-1 and hPDL-2 as determined by ELISA. The anti-PD-1 antibodies nivolumab (“NV1”) and pembrolizumab (“PM1”) were used as positive controls.

FIG. 8 is a graph depicting the ability of various anti-hPD-1 antibodies of the disclosure referred to herein as A1.5, Ba2, Bb2, C1.1, and D4 to inhibit binding between hPD-1 and hPDL-1 as determined by ELISA. The anti-PD-1 antibodies nivolumab (“NV1”) and pembrolizumab (“PM1”) were used as positive controls.

FIG. 9 is a graph depicting the ability of various anti-hPD-1 antibodies of the disclosure referred to herein as A1.5, Ba2, Bb2, C1.1, and D4 to inhibit binding between hPD-1 and hPDL-2 as determined by ELISA. The anti-PD-1 antibodies nivolumab (“NV1”) and pembrolizumab (“PM1”) were used as positive controls.

FIG. 10 is a graph depicting that the anti-PD-1 antibody referred to herein as A1.5 specifically binds hPD-1 as determined by ELISA. The A1.5 anti-PD-1 antibody was tested against a panel of human and mouse proteins.

FIGS. 11A and 11B are a series of graphs depicting the ability of the anti-PD-1 antibody A1.5 to block Fab fragments of the anti-PD-1 antibodies nivolumab (“NV1”) and pembrolizumab (“PM1”) from binding to PD-1. These graphs also demonstrate that the anti-PD-1 antibody A1.5 binds a distinct epitope from either NV1 and/or PM1.

FIG. 12 is a graph demonstrating that the anti-PD-1 antibody A1.5 has a potency similar to that of nivolumab (“NV1”) and pembrolizumab (“PM1”) measured in a T-cell restimulation assay. In FIG. 12, PMBCs were treated as follows: PBMCs: CMV+HemaCare Donor C were plated at 2×10⁵ cells/well; stimulated with 5 μg/mL CMV lysate+/−anti-PD-1 or isotype control; IFN-γ ELISA assay performed on day 4 supernatant.

FIG. 13 is a series of graphs demonstrating that the anti-PD-1 antibodies A1.5 and Ba2 bind monomeric human PD-1 (hPD1) with similar or higher affinity than the anti-PD-1 antibodies nivolumab (“Nivo”) and pembrolizumab (“Pembro”).

FIG. 14 is a series of graphs depicting the binding to human PD-1 by anti-PD-1 antibody A1.4 and by various activatable anti-PD-1 antibodies that include the various masking moieties referred to herein as PD01 (SEQ ID NO: 66), PD02 (SEQ ID NO: 67), PD03 (SEQ ID NO: 68), PD08 (SEQ ID NO: 73), PD09 (SEQ ID NO: 74), and PD10 (SEQ ID NO: 75) as determined by ELISA.

FIG. 15 is a series of graphs depicting the binding to hPD-1 by anti-PD-1 antibody A1.5 and by various activatable anti-PD-1 antibodies that include the anti-PD-1 antibody A1.5 and the masking moieties referred to herein as PD01 (SEQ ID NO: 66), PD02 (SEQ ID NO: 67), PD03 (SEQ ID NO: 68), PD05 (SEQ ID NO: 70), PD06 (SEQ ID NO: 71) (Plate 1); PD08 (SEQ ID NO: 73), PD09 (SEQ ID NO: 74), PD10 (SEQ ID NO: 75), PD11 (SEQ ID NO: 76), PD12 (SEQ ID NO: 77) (Plate 2); PD13 (SEQ ID NO: 78), PD14 (SEQ ID NO: 79), PD15 (SEQ ID NO: 80), PD16 (SEQ ID NO: 81), PD17 (SEQ ID NO: 82) (Plate 3); PD18 (SEQ ID NO: 83), PD19 (SEQ ID NO: 84), PD20 (SEQ ID NO: 85), PD21 (SEQ ID NO: 86), PD22 (SEQ ID NO: 87) (Plate 4); and PD23 (SEQ ID NO: 88), PD24 (SEQ ID NO: 89) (Plate 5).

FIG. 16 is a graph depicting the binding to hPD-1 by anti-PD-1 antibody A1.5 and various activatable anti-PD-1 antibodies that include the anti-PD-1 antibody A1.5, the cleavable moiety referred to herein as 3001, which includes the sequence AVGLLAPPGGLSGRSDNH (SEQ ID NO: 318), and the masking moieties referred to herein as PD02, PD12, and PD16.

FIG. 17 is a series of graphs depicting the binding to hPD-1 by the anti-PD-1 antibody A1.5 and by various activatable anti-PD-1 antibodies that include the anti-PD-1 antibody A1.5, the cleavable moiety referred to herein as 2001, which includes the sequence ISSGLLSGRSDNH (SEQ ID NO: 214), and the masking moieties referred to herein as PD05 (SEQ ID NO: 70), PD06 (SEQ ID NO: 71), PD09 (SEQ ID NO: 74) (upper left panel); PD12 (SEQ ID NO: 77), PD23 (SEQ ID NO: 88), PD25 (SEQ ID NO: 90) (upper right panel); PD26 (SEQ ID NO: 91), PD27 (SEQ ID NO: 92), PD28 (SEQ ID NO: 93) (lower left panel); and PD30 (SEQ ID NO: 95), PD33 (SEQ ID NO: 98), PD35 (SEQ ID NO: 100) (lower right panel).

FIG. 18 is a series of graphs depicting the binding to hPD-1 by the anti-PD-1 antibody A1.5 and by various activatable anti-PD-1 antibodies that include the anti-PD-1 antibody A1.5 and the masking moieties referred to herein as PD34 (SEQ ID NO: 99), PD06 (SEQ ID NO: 71), PD09 (SEQ ID NO: 74) (upper left panel); PD34 (SEQ ID NO: 99), PD12 (SEQ ID NO: 77), PD17 (SEQ ID NO: 82) (upper right panel); PD34 (SEQ ID NO: 99), PD19 (SEQ ID NO: 84), PD25 (SEQ ID NO: 90) (lower left panel); and PD34 (SEQ ID NO: 99), PD26 (SEQ ID NO: 91), PD28 (SEQ ID NO: 93) (lower right panel).

FIG. 19 is a graph depicting the ability of anti-PD1 A1.5 activatable antibodies of the disclosure to increase CMV-stimulated IFN-gamma secretion as compared to control hIgG4, but with decreased potency relative to anti-PD1 A1.5 parental antibody. The tested activatable antibodies include the anti-PD-1 antibody A1.5, the cleavable moiety referred to herein as 2001, and the masking moieties referred to herein as PD06, PD05, PD11, PD12, PD14, and PD19. In FIG. 19, PMBCs were treated as follows: PBMCs: CMV+ HemaCare Donor C were plated at 2×10⁵ cells/well; stimulated with 5 μg/mL CMV lysate+/−anti-PD-1 or isotype control; IFN-γ ELISA assay performed on day 4 supernatant.

FIGS. 20A and 20B are a series of graphs depicting the ability of the anti-PD-1 antibody referred to herein as J43v2 (Heavy Chain (HC): SEQ ID NO: 546; Light Chain (LC): SEQ ID NO: 543) to block murine PD-L1 (mPD-L1) and murine PD-L2 (mPD-L2) from binding to cells expressing murine PD-1 (mPD-1) with single digit nM EC₅₀ values.

FIG. 21 is a graph depicting the dose-dependent induction of diabetes in NOD mice by the antibody referred to herein as J43v2 m2a (HC: SEQ ID NO: 546; LC: SEQ ID NO: 543).

FIG. 22 is a series of graphs depicting the binding to hPD-1 by anti-PD-1 antibody J43v2 m2a and by various activatable anti-PD-1 antibodies that include the anti-PD-1 antibody J43v2 m2a and the masking moieties referred to herein as MP01 (SEQ ID NO: 384), MP02 (SEQ ID NO: 385), MP03 (SEQ ID NO: 386), MP04 (SEQ ID NO: 387), MP05 (SEQ ID NO: 388) (upper left panel); MP06 (SEQ ID NO: 389), MP07 (SEQ ID NO: 390), MP08 (SEQ ID NO: 391), MP09 (SEQ ID NO: 392), MP10, also referred to herein as MP010 (SEQ ID NO: 393) (upper right panel); MP11, also referred to herein as MP011 (SEQ ID NO: 394), MP12, also referred to herein as MP012 (SEQ ID NO: 395), MP13, also referred to herein as MP013 (SEQ ID NO: 396), MP14, also referred to herein as MP014 (SEQ ID NO: 397) (bottom panel).

FIG. 23 is a graph depicting the binding to hPD-1 by the anti-PD-1 antibody J43v2 m2a and by various activatable anti-PD-1 antibodies that include the anti-PD-1 antibody J43v2 m2a, the cleavable moiety referred to herein as 2001, and the masking moieties referred to herein as MP5-2 (SEQ ID NO: 565), MP7-1 (SEQ ID NO: 558), MP7-5 (SEQ ID NO: 562), MP8-2 (SEQ ID NO: 549) (upper panel); and MP7-1 (SEQ ID NO: 558), MP8-9 (SEQ ID NO: 556), MP8-8 (SEQ ID NO: 555) (lower panel).

FIG. 24 is a graph depicting that the anti-PD-1 J43v2 antibody induced diabetes in NOD mice with increased frequency as dosage increased and that anti-PD-1 J43v2 activatable antibodies exhibited reduced diabetes compared to antibodies at similar doses.

FIGS. 25A and 25B are a series of graphs depicting the binding to hPD-1 by the anti-PD-1 antibody A1.5 and by of various activatable antibodies of the disclosure comprising the anti-PD1 antibody A1.5, the masking moiety referred to herein as PD34 (SEQ ID NO: 99), and for FIG. 25A, the substrates referred to herein as 2006 (SEQ ID NO: 1095), 2007 (SEQ ID NO: 1096), 2008 (SEQ ID NO: 1097), 2009 (SEQ ID NO: 1098), and 2001 (SEQ ID NO: 214), and for FIG. 25B, the substrates referred to herein as 2001 (SEQ ID NO: 214), 2008 (SEQ ID NO: 1097), 2012 (SEQ ID NO: 1101), 2011 (SEQ ID NO: 1100), and 2003 (SEQ ID NO: 1092).

FIGS. 26A, 26B, and 26C are a series of graphs depicting the binding to hPD-1 by the anti-PD-1 antibody nivolumab (NV1) and by of various activatable antibodies of the disclosure comprising the anti-PD1 antibody NV1, the substrate referred to herein as 2001 (SEQ ID NO: 214), and the masking moieties referred to herein NV01 (SEQ ID NO: 1206), NV02 (SEQ ID NO: 1207), NV03 (SEQ ID NO: 1208), NV04 (SEQ ID NO: 1209), NV05 (SEQ ID NO: 1210), NV06 (SEQ ID NO: 1211), NV07 (SEQ ID NO: 1212), NV08 (SEQ ID NO: 1213), NV09 (SEQ ID NO: 1214), NV10 (SEQ ID NO: 1215), NV11 (SEQ ID NO: 1216), and NV12 (SEQ ID NO: 1217).

FIGS. 27A, 27B, and 27C are a series of graphs depicting the binding to hPD-1 by the anti-PD-1 antibody pembrolizumab (PM1) and by various activatable antibodies of the disclosure comprising the anti-PD1 antibody PM1, the substrate referred to herein as 2001 (SEQ ID NO: 214), and the masking moieties referred to herein PM01 (SEQ ID NO: 1351), PM02 (SEQ ID NO: 1352), PM03 (SEQ ID NO: 1353), PM04 (SEQ ID NO: 1354), PM05 (SEQ ID NO: 1355), PM06 (SEQ ID NO: 1356), PM07 (SEQ ID NO: 1357), PM08 (SEQ ID NO: 1358), PM09 (SEQ ID NO: 1359), PM10 (SEQ ID NO: 1360), PM11 (SEQ ID NO: 1361), and PM12 (SEQ ID NO: 1362).

FIG. 28 is a graph depicting the masking moiety specificity for various activatable antibodies of the disclosure that comprise the anti-PD-1 A1.5 (PD) antibody, the NV1 (NV) antibody, and the PM1 (PM) antibody and a variety of mask and substrate combinations.

FIGS. 29A and 29B are a series of graphs depicting the binding to PD1 by the IgG2a effector negative (EN) anti-PD1 antibody J43 (J43 m2a EN) and by various activatable antibodies of the disclosure comprising the IgG2a effector negative (EN) anti-PD1 antibody J43 (J43 m2a EN) and a variety of mask and substrate combinations.

FIG. 30 is a graph depicting that the anti-PD-1 J43m2a EN antibody induced diabetes in NOD mice with increased frequency as dosage increased and that anti-PD-1 J43m2a EN activatable antibodies exhibited reduced diabetes compared to antibodies at similar doses.

FIGS. 31A and 31B are a series of graphs depicting that anti-PD1 activatable antibodies of the disclosure MP7-1 2001 m2a EN and MP8-2 2001 m2a EN inhibit the growth of MC38 syngeneic tumors in a manner similar to positive control anti-PD1 antibody J43 m2a EN both as single agents (FIG. 31A) and in combination with the commercially available anti-CTLA4 antibody 9D9 mIgG2b (FIG. 31B).

FIGS. 32A, 32B, 32C, 32D, and 32E are a series of graphs depicting that the anti-human PD1 antibody referred to herein as A1.5 Ab (i.e., VH of SEQ ID NO: 21 and VL of SEQ ID NO: 47) blocks PD-L1/PD-L2 binding to PD1 and potently activates T cells in an antigen recall assay. FIG. 31A shows the binding of the anti-human PD-1 A1.5 Ab to immobilized human PD1 as detected by standard plate ELISA, and FIG. 31B shows the binding of A1.5 Ab to cynomolgus PD1 as detected by ELISA. FIG. 32C depicts the inhibition of biotinylated human PD-L1 to immobilized PD1 by A1.5 Ab as determined by ELISA, and FIG. 32D depicts the inhibition of biotinylated human PD-L2 to immobilized PD1 as determined by ELISA. FIG. 32E demonstrations that the A1.5 Ab enhances IFN-γ production in a CMV T cell restimulation assay.

FIGS. 33A and 33B are a series of graphs depicting that the activatable anti-PD1 antibody referred to herein as A1.5 PD34 2001 (i.e., VH of SEQ ID NO: 21, VL of SEQ ID NO: 47, masking moiety of SEQ ID NO: 99, and cleavable moiety of SEQ ID NO: 214) binds human PD1 with decreased affinity relative to the parental PD-1 Ab, i.e., A1.5 Ab (FIG. 33A), and the A1.5 PD34 2001 activatable antibody shows functional masking in a CMV T cell antigen recall assay (FIG. 33B).

FIG. 34 is a graph and a table depicting the results of pharmacokinetic (PK) analysis of plasma samples from cynomolgus monkeys after dosing with a single IV bolus dose of either the A1.5 antibody or the activatable antibody referred to herein as A1.5 PD34 2001 at 1 mg/kg or at 5 mg/kg. Mean PK parameters shown for both A1.5 and A1.5 PD34 2001.

FIG. 35 is a graph depicting that the combination of 10 mg/kg anti-PD-1 J43 antibody plus 10 mg/kg anti-CTLA-4 antibody dosed on days 0, 4 and 7 induced diabetes in 50% of NOD mice by day eleven while the same dosing schedule of activatable anti-PD-1 J43 antibodies with CTLA-4 antibody resulted in no induction of diabetes to day eighteen.

FIG. 36 is a graph depicting that the anti-PD1 activatable antibodies MP8-2 2012 m2a EN and MP8-2 2011 m2a EN inhibit the growth of MC38 syngeneic tumors similar to positive control anti-PD1 antibody J43 m2a EN, both as single agents and in combination with anti-CTLA4 antibody 9D9 mIgG2b.

FIG. 37 is a series of graphs depicting that combination CTLA4 antibody and J43 activatable antibody treatment protects mice against rechallenge with MC38 tumor cells.

FIG. 38 is a graph depicting that an anti-PD-1 antibody administered alone or in combination with an anti-CTLA4 antibody induced diabetes in NOD mice, whereas an anti-PD-1 activatable antibody as a single agent or in combination with an anti-CTLA4 antibody resulted in no induction of diabetes to day fifteen.

FIG. 39 is a graph demonstrating that anti-PD-1 activatable A1.5 and activatable nivolumab antibodies of the embodiments enhance IFN-gamma production in a CMV T cell restimulation assay as compared to a control antibody, but not to the extent effected by anti-PD-1 antibody A1.5 or nivolumab.

FIG. 40 is a graph demonstrating that an anti-PD-1 activatable pembrolizumab antibody of the embodiments enhances IFN-gamma production in a CMV T cell restimulation assay as compared to a control antibody, but not to the extent effected by anti-PD-1 antibody A1.5 or pembrolizumab.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides monoclonal antibodies (mAbs), activatable antibodies, and antigen-binding fragments thereof that specifically bind programmed cell death protein 1 (PD-1), also known as CD279. The use of the term “PD-1” is intended to cover any variation thereof, such as, by way of non-limiting example, PD1 and/or PD 1, all variations are used herein interchangeably. Aberrant expression and/or activity of PD-1 and PD-1-related signaling has been implicated in the pathogenesis of many diseases and disorders, such as cancer.

PD-1, a cell surface receptor that belongs to the immunoglobulin superfamily, is expressed on T cells and pro-B cells. PD-1 is known to bind two ligands, PD-L1 and PD-L2. PD-1 is expressed on the surface of activated T cells, and the interaction between PD-1 and PD-L1 and/or PD-L2 functions as an immune checkpoint, as the binding of PD-L1 or PD-L2 to PD-1 inactivates the T cell. Thus, PD-1 plays a role in down-regulating the immune system by preventing the activation of T-cells, which, in turn, reduces autoimmunity and promotes self-tolerance.

The anti-PD-1 monoclonal antibodies and activated activatable anti-PD-1 antibodies of the disclosure bind and neutralize or otherwise inhibit the ability of PD-1 to bind or otherwise interact with PD-L1 and/or PD-L2. The anti-PD-1 monoclonal antibodies and activated activatable anti-PD-1 antibodies of the disclosure bind and neutralize or otherwise inhibit at least one biological activity of PD-1. For example, the anti-PD-1 monoclonal antibodies and activated activatable anti-PD-1 antibodies of the disclosure bind PD-1 and block or otherwise inhibit ligand activation of PD-1 on activated T cells. In contrast to traditional chemotherapies and other targeted anti-cancer therapies, which exert their effects by direct cytotoxic or tumor growth inhibition, the monoclonal antibodies and activatable antibodies of the disclosure block a negative regulator of T-cell activation and response, thereby allowing the immune system to attack the tumor.

The activatable anti-PD-1 antibodies of the disclosure include an antibody or antigen-binding fragment thereof that specifically binds PD-1 coupled to a masking moiety (MM), such that coupling of the MM reduces the ability of the antibody or antigen-binding fragment thereof to bind PD-1. The MM is coupled via a cleavable moiety (CM) that includes a sequence that functions as a substrate for a protease.

The activatable anti-PD-1 antibodies of the disclosure are activated when the cleavable moiety is cleaved by a protease. For example, the protease is produced by a tumor that is in proximity to T cells that express PD-1. In some embodiments, the protease is produced by a tumor that is co-localized with T cells that express PD-1. In the activated, i.e., cleaved state, the activatable anti-PD-1 antibodies of the disclosure bind PD-1 expressed on a T cell surface.

The activatable anti-PD-1 antibodies are used in methods of treating, preventing, delaying the progression of, ameliorating and/or alleviating a symptom of a disease or disorder associated with binding of a ligand selected from the group consisting of PD-L1 and PD-L2 to PD-1 on a T cell. Such ligands are often found on tumor cells and other immune cells. Binding of such ligands to PD-1 can lead to immune suppression, such as suppression of T cell activity, which is reduced by anti-PD1 antibodies and activatable anti-PD-1 antibodies of the disclosure. For example, the activatable anti-PD-1 antibodies are used in methods of treating, preventing, delaying the progression of, ameliorating and/or alleviating a symptom of a cancer or other neoplastic condition.

Exemplary activatable anti-PD-1 antibodies of the invention include, for example, activatable antibodies that include a heavy chain and a light chain that are, or are derived from, antibodies described in the Examples, for example in Examples 1, 2, 8, 9, 14, and 15.

In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 572, 574, 576, 578, 580, 582, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1030, 1032, 1034, 1036, 1039, 1041-1090, 1113-1120, 1123, 1124, 1127, 1128, 1131, 1132, 1134, 1135, 1138, 1139, 1143, 1144, 1147, 1148, 1151, 1152, 1155, 1156, 1159, 1160, 1163, 1164, 1167, 1168, 1170, 1171, 1174, 1175, 1178, 1179, 1182, 1183, 1186, 1187, 1190, 1191, 1194, 1195, 1198, 1199, 1202, 1203, 2055, 2054, 2057, 2056, 2059, and 2058.

In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 572, 574, 576, 578, 580, 582, 584, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029, 1030, 1032, 1034, 1036, 1039, 1041-1090, 1113-1120, 1123, 1127, 1131, 1134, 1138, 1144, 1148, 1152, 1156, 1160, 1164, 1168, 1170, 1174, 1178, 1182, 1186, 1190, 1194, 1198, 1203, 2055, 2054, 2057, 2056, 2059, and 2058.

In some embodiments, the light chain comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. In some embodiments, the light chain comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 584, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, and 997.

In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain that comprises or is derived from an amino acid sequence selected from the group consisting of 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 572, 574, 576, 578, 580, 582, 1029, 1120, 1124, 1128, 1132, 1135, 1139, 1143, 1147, 1151, 1155, 1159, 1163, 1167, 1171, 1175, 1179, 1183, 1187, 1191, 1195, 1199, 1203, 2055, 2054, 2057, 2056, 2059, and 2058.

In some embodiments, the light chain comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 572, 574, 576, 578, 580, 582, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029, 1030, 1032, 1034, 1036, 1039, 1041-1090, 1113-1120, 1123, 1127, 1131, 1134, 1138, 1144, 1148, 1152, 1156, 1160, 1164, 1168, 1170, 1174, 1178, 1182, 1186, 1190, 1194, 1198, 1203, 2055, 2054, 2057, 2056, 2059, and 2058.

In some embodiments, the activatable anti-PD-1 antibody includes a light chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 572, 574, 576, 578, 580, 582, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 1027, 1028, 1029, 1030, 1032, 1034, 1036, 1039, 1041-1076, 1113-1120, 1123, 1124, 1127, 1128, 1131, 1132, 1134, 1135, 1138, 1139, 1143, 1144, 1147, 1148, 1151, 1152, 1155, 1156, 1159, 1160, 1163, 1164, 1167, 1168, 1170, 1171, 1174, 1175, 1178, 1179, 1182, 1183, 1186, 1187, 1190, 1191, 1194, 1195, 1198, 1199, 1202, 12032055, 2054, 2057, 2056, 2059, and 2058.

In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 33, 35, and 37, and a light chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 1028, 1029, 1041-1076, 1138, 1139, 1143, 1144, 1147, 1148, 1151, 1152, 1155, 1156, 1159, 1160, 1163, 1164, 1167, 1168, 1170, 1171, 1174, 1175, 1178, 1179, 1182, 1183, 1186, 1187, 1190, 1191, 1194, 1195, 1198, 1199, 1202, 1203, 2055, 2054, 2057, 2056, 2059, and 2058.

In some embodiments, the light chain comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 1028, 1041-1076, 1138, 1144, 1148, 1152, 1156, 1160, 1164, 1168, 1170, 1174, 1178, 1182, 1186, 1190, 1194, 1198, 1202, 2055, 2057, and 2059.

In some embodiments, the light chain comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 1029, 1139, 1143, 1147, 1151, 1155, 1159, 1163, 1167, 1171, 1175, 1179, 1183, 1187, 1191, 1195, 1199, 1203, 2054, 2056, and 2058.

In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 546 and a light chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 572, 574, 576, 578, 580, 582, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1030, 1032, 1034, 1036, 1039, 1077-1090, 1113-1120, 1123, 1124, 1127, 1128, 1131, 1132, 1134, and 1135.

In some embodiments, the activatable anti-PD-1 antibody includes a light chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NOS: 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1030, 1032, 1034, 1036, 1039, 1070-1090, 1119, 1123, 1127, 1131, and 1134.

In some embodiments, the activatable anti-PD-1 antibody includes a light chain that comprises or is derived from an amino acid sequence selected from the group consisting of SEQ ID NOS: 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 572, 574, 576, 578, 580, 582, 1120, 1124, 1128, 1132, and 1135.

In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain that comprises or is derived from a heavy chain amino acid sequence shown in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a light chain that comprises or is derived from a light chain amino acid sequence shown in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a heavy chain that comprises or is derived from a heavy chain amino acid sequence shown in Table 7, and a light chain that comprises or is derived from a light chain amino acid sequence shown in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group A in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group B in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group C in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group D in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group E in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group F in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group H in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group I in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group J in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group K in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group L in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group M in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain variable region sequence and the light chain variable region sequence as shown in Group N in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain variable region sequence and the light chain variable region sequence as shown in Group O in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group P in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of a heavy chain variable region sequence and a light chain variable region sequence from the combinations shown in Group Q in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the complementarity determining region (CDR) sequences of a heavy chain sequence from the heavy chain sequences shown in Group A in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group A in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group A in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group A in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of CDRs of a heavy chain sequence from the heavy chain sequences shown in Group B in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group B in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group B in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group B in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group C in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group C in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group C in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group C in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group D in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group D in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group D in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group D in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group E in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group E in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group E in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group E in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group F in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group F in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group F in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group F in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group G in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group G in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group G in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group G in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group H in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group H in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group H in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group H in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group I in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group I in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group I in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group I in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group J in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group J in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group J in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group J in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group K in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group K in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group K in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group K in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group L in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group L in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group L in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group L in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group M in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group M in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group M in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group M in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group N in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group N in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group N in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group N in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group O in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group O in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group O in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group O in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group P in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group P in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group P in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group P in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group Q in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group Q in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group Q in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group Q in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group R in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group R in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group R in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group R in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group S in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group S in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group S in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group S in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group T in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group T in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group T in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group T in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group U in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group U in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group U in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group U in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group V in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 619 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1856 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 619 and the light chain sequence of SEQ ID NO: 1856 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1846 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1858 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1846 and the light chain sequence of SEQ ID NO: 1858 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1843 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1859 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1843 and the light chain sequence of SEQ ID NO: 1859 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1847 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1847 and the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1848 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1860 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1848 and the light chain sequence of SEQ ID NO: 1860 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1844 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1861 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1844 and the light chain sequence of SEQ ID NO: 1861 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1841 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1841 and the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1842 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1842 and the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1845 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1845 and the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1835 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1857 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1835 and the light chain sequence of SEQ ID NO: 1857 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1836 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1862 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1836 and the light chain sequence of SEQ ID NO: 1862 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1837 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1863 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1837 and the light chain sequence of SEQ ID NO: 1863 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1838 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1864 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1838 and the light chain sequence of SEQ ID NO: 1864 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1838 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1865 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1838 and the light chain sequence of SEQ ID NO: 1865 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1839 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1864 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1839 and the light chain sequence of SEQ ID NO: 1864 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1839 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1865 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1839 and the light chain sequence of SEQ ID NO: 1865 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1840 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1866 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1840 and the light chain sequence of SEQ ID NO: 1866 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1841 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1867 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1841 and the light chain sequence of SEQ ID NO: 1867 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1841 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1868 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1841 and the light chain sequence of SEQ ID NO: 1868 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1849 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1858 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1849 and the light chain sequence of SEQ ID NO: 1858 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1853 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1859 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1853 and the light chain sequence of SEQ ID NO: 1859 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1869 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1869 and the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1850 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1860 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1850 and the light chain sequence of SEQ ID NO: 1860 as shown in Group V in Table 7.

n some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1851 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 1861 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1851 and the light chain sequence of SEQ ID NO: 1861 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1852 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1852 and the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1854 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1854 and the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of the heavy chain sequence of SEQ ID NO: 1855 and a combination of the CDRs of the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the heavy chain sequence of SEQ ID NO: 1855 and the light chain sequence of SEQ ID NO: 737 as shown in Group V in Table 7.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group W in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a light chain sequence from the light chain sequences shown in Group W in Table 7. In some embodiments, the activatable anti-PD-1 antibody includes a combination of the CDRs of a heavy chain sequence from the heavy chain sequences shown in Group W in Table 7 and the CDRs of a light chain sequence from the heavy chain sequences shown in Group W in Table 7.

TABLE 7 Variable Heavy Chain Region (VH) and Variable Light Chain Region (VL) Sequences for Activatable Antibodies that Bind PD-1 Group A VH QVQLVESGGDVVQPGGSLRLSCAASGVAFSNYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISR DNSKNMLYLQMNSLRAEDTAMYYCARNDDYWGQGTLVTVSS (SEQ ID NO: 615) VH QVQLVESGGDVVQPGRSLRLSCAASGLTFTNYGFHWVRQAPGKGLEWVAVIWYDGSKKYYADSVKGRFTISR DNSKNTLYLQMNNLRAEDTAVYYCATGDDYWGQGTLVTVSS (SEQ ID NO: 617) VH QVYLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVALIWYDGSNKYYADSVKGRFTISR DNSKNTLYLQMTSLRVEDTAVYYCASNVDHWGQGTLVTVSS (SEQ ID NO: 618) VH QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISR DNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS (SEQ ID NO: 1346) VH QLQLQESGPGLVKPSETLSLTCTVSGGSLSRSSFFWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVYYCVRDYDILTGDEDYWGQGTLVTVSS (SEQ ID NO: 620) VH QVQLVESGGGVVQPGRSLRLSCTTSGITFSSYGFHWVRQAPGKGLEWVAVIWYDGSKKYYADSVKGRFTLSR DDSKNTLYLQMNSLRAEDTAVYYCVTGDDYWGQGTLVTVSS (SEQ ID NO: 621) VH QLQLQESGPGLVKPSETLSLTCSVSGGSLSRSSYFWGWIRQPPGKGLEWIASIFYSGETYFNPSLKSRVTIS VDTSRNQFSLKLSSVTAADTAVYYCARDYDILTGDEDYWGQGTLVTVSS (SEQ ID NO: 623) VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLIIYDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK (SEQ ID NO: 624) VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDTSNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK (SEQ ID NO: 625) VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK (SEQ ID NO: 626) VL DIQMTQSPSSLSASVGDRVSITCRASQGISSWLAWYQQKPEKAPKSLIYAASNLRSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQYYSYPRTFGQGTKVEIK (SEQ ID NO: 628) VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK (SEQ ID NO: 629) VL DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQYYSYPRTFGQGTKVEIK (SEQ ID NO: 630) Group B VH QVQLQQPGAELVKPGASVKLSCKASGYTFTSYYLYWMKQRPGQGLEWIGGVNPSNGGTNFSEKFKSKATLTV DKSSSTAYMQLSSLTSEDSAVYYCTRRDSNYDGGFDYWGQGTTLTVSSAK (SEQ ID NO: 631) VH QVQLQQPGAELVKPGTSVKLSCKASGYTFTNYYMYWVKQRPGQGLEWIGGINPSNGGTNFNEKFKNKATLTV DSSSSTTYMQLSSLTSEDSAVYYCTRRDYRFDMGFDYWGQGTTLTVSSAK (SEQ ID NO: 632) VH MDWTWSILFLVAAPTGAHSQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINP SNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 633) VH QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTT DSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 634) VH EVQLVLSGGGFVQPGGSLKLSCAASGFTFSSYAMSWVRQNPERRLVWVATITGGGRNTYYPDSVKGRFTISR DNAKNTLYLQMSSLRSEDTAMYYCTRQGYDGYTWFAYWGQGTLVTVS (627) VL DIVLTQSPTSLAVSLGQRATISCRASKSVSTSGFSYLHWYQQKPGQPPKLLIFLASNLESGVPARFSGSGSG TDFTLNIHPVEEEDAATYYCQHSWELPLTFGAGTKLELK (SEQ ID NO: 635) VL DIVLTQSPASLAVSLGQRAAISCRASKGVSTSGYSYLHWYQQKPGQSPKLLIYLASYLESGVPARFSGSGSG TDFTLNIHPVEEEDAATYYCQHSRDLPLTFGTGTKLELK (SEQ ID NO: 636) VL MAPVQLLGLLVLFLPAMRCEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIY LASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK (SEQ ID NO: 637) VL EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSG TDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK (SEQ ID NO: 638) VL MAPVQLLGLLVLFLPAMRCEIVLTQSPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIY LASYLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSRDLPLTFGQGTKLEIK (SEQ ID NO: 639) VL EIVLTQSPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLESGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCQHSRDLPLTFGQGTKLEIK (SEQ ID NO: 640) VL MAPVQLLGLLVLFLPAMRCDIVMTQTPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIY LASYLESGVPDRFSGSGSGTAFTLKISRVEAEDVGLYYCQHSRDLPLTFGQGTKLEIK (SEQ ID NO: 641) VL DIVMTQTPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLESGVPDRFSGSGSG TAFTLKISRVEAEDVGLYYCQHSRDLPLTFGQGTKLEIK (SEQ ID NO: 642) VL DIVLTQSPTSLAVSLGQRATISCRASESVDNSGISFMNWFQQKPGQPPKLLIYAASNPGSGVPARFSGSGSG TDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTELEIKR (SEQ ID NO: 725) HC MAVLGLLFCLVTFPSCVLSQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINP SNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 643) HC QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTT DSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK (SEQ ID NO: 644) HC MAVLGLLFCLVTFPSCVLSQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINP SNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 645) HC QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTT DSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK (SEQ ID NO: 646) LC MAPVQLLGLLVLFLPAMRCEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIY LASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 647) LC EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSG TDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC (SEQ ID NO: 648) LC MAPVQLLGLLVLFLPAMRCEIVLTQSPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIY LASYLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSRDLPLTFGQGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 649) LC EIVLTQSPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLESGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCQHSRDLPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC (SEQ ID NO: 650) LC MAPVQLLGLLVLFLPAMRCDIVMTQTPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIY LASYLESGVPDRFSGSGSGTAFTLKISRVEAEDVGLYYCQHSRDLPLTFGQGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 651) LC DIVMTQTPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLESGVPDRFSGSGSG TAFTLKISRVEAEDVGLYYCQHSRDLPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC (SEQ ID NO: 652) Group C VH DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYINYSGSTSYNPSLKSRISITR DTSKNQFFLQLNSVTTEDTATYYCARWIGSSAWYFDVWGAGTTVTV (SEQ ID NO: 726) VL DVLMTQTPLSLPVSLGDQASISCRSGQNIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFFGVPDRISGSGS GTDFTLKISRVEAEDLGVYFCFQGSHVPFTFGSGTKLEIK (SEQ ID NO: 727) VH QVQLQQPGAELVKPGASVKLSCKASGYTFTTYYLYWVRQRPGQGLEWIGGINPSNGGTNFNEKFKSKATLTV DKSSSTAYMQLNSLTSEDSAVYYCTRRDYRYDRGFDYWGQGTSVTV (SEQ ID NO: 728) VL DIVLTQSPASLAVSLGQRATISCRASKSVSTSGFNYIHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSG TDFTLNIHPVEDEDAATYYCQHSRELPLTFGAGTKLEIK (SEQ ID NO: 729) VH QVQLQQSGAELVKPGASVKMSCKAFGYTFTTYPIEWMKQNHGKSLEWIGNFHPYNDDTKYNEKFKGKAKLTV EKSSTTVYLELSRLTSDDSAVYYCARENYGSHGGFVYWGQGTLVTV (SEQ ID NO: 730) VL ENVLTQSPAIMSASPGEKVTMTCRASSSVISSYLHWYQQKSGASPKLWIYSTSNLASGVPDRFSGSGSGTSY SLTISSVEAEDAATYYCQQYNGYPLTFGAGTKLEIK (SEQ ID NO: 731) Group D LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQ VQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPAN TSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 732) Group E VH EVQLVQSGAEVKKPGASVKVSCKASGYRFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDADYSSGSGYWGQGTLVTVSS (SEQ ID NO: 733) VH QVQLVQSGAEVKKPGASVRVSCKASGYTLTSYYIHWVRQAPGQGLEWMGIINPRGATISYAQKFQGRVTMTR DTSTSTVYMELRNLKSEDTALYYCATAGIYGFDFDYWGRGTLVTVSS (SEQ ID NO: 734) VH QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGAYYWSWIRQHPGKGLEWIGYIYYNGNTYYNPSLRSLVTIS VDASKNQFSLKLSSVTAADTAVYYCARASDYVWGGYRYMDAFDIWGRGTLITVSS (SEQ ID NO: 735) VH GAHSEVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYWCDRMSWVRQAPGKGLEWVSAISGSGGSTYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKENWGSYFDLWGQGTTVTVSS (SEQ ID NO: 736) VL SYELTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQKPGQAPVMVIYKDTERPSGIPERFSGSSSGTKVTL TISGVQAEDEADYYCQSADNSITYRVFGGGTKVTVL (SEQ ID NO: 737) VL QSALTQPASVSGSPGQSITISCTGTSNDVGGYNYVSWYQHHPGKAPKLIIYDVTNRPSGVSDRFSGSKSGNT ASLTISGLLAEDEGDYYCSSYTIVTNFEVLFGGGTKLTV (SEQ ID NO: 738) VL QSVLTQPPSASGTPGQRVTISCSGSNSNIGSNSVNWYQQLPGTAPKLLIYGNNQRPSGVPDRFSGSKSGTSA SLAISGLQSENEADYYCAAWDDSLNGPVFGRGTKVTVLGE (SEQ ID NO: 739) VL GVHSDIVMTQSPSTLSASVGDRVTITCRASQGISSWLAWYQQKPGRAPKVLIYKASTLESGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKLEIKR (SEQ ID NO: 740) Group F VH QVQLVQSGHEVKQPGASVKMSCKASGYSFTSSWIHWVKQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATLTA DKSTSTAYMELSSLRSEDTAVYYCARWRDSSGYHAMDYWGQGTLVTVSS (SEQ ID NO: 741) VH QVQLVQSGHEVKQPGASVKMSCKASGYSFTSSWIHWVRQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATLTA DKSTSTAYMELSSLRSEDTAVYYCARWRDSSGYHAMDYWGQGTLVTVSS (SEQ ID NO: 742) VL EIVLTQSPATLSLSPGQRLTISCRASQSVSTSGYSYMHWYQQKPDQSPKLLIKFGSNLESGIPARFSGSGSG TDFTLTISSLEPEDFATYYCQHSWEIPYTFGQGTKLEIK (SEQ ID NO: 743) VL DIVLTQSPATLSLSPGQRLTISCRASQSVSTSGYSYMHWYQQKPDQSPKLLIKFGSNLESGIPARFSGSGSG TDFTLTISSLEPEDFAVYYCQHSWEIPYTFGQGTKLEIK (SEQ ID NO: 744) Group H VH QGQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPIHGLEWIGVIESETGGTAYNQKFKGRVTITA DKSTSTAYMELSSLRSEDTAVYYCAREGITTVATTYYWYFDVWGQGTTVTVSS (SEQ ID NO: 745) VL DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIK (SEQ ID NO: 746) Group I VH MGLGLQWVFFVALLKGVHCEVRLLESGGGLVKPEGSLKLSCVASGFTFSDYFMSWVRQAPGKGLEWVAHIYT KSYNYATYYSGSVKGRFTISRDDSRSMVYLQMNNLRTEDTATYYCTRDGSGYPSLDFWGQGTQVTVSSATTT APSVYPLAPACDSTTKS (SEQ ID NO: 747) VL YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLT IRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYP GSATVTWKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSLSPAECL (SEQ ID NO: 748) Bs METDTLLLWVLLLWVPGSTGDAAQPARRARRTKLGTELGSPGLQEFEVRLLESGGGLVKPEGSLKLSCVASG Ab FTFSDYFMSWVRQAPGKGLEWVAHIYTKSYNYATYYSGSVKGRFTISRDDSRSMVYLQMNNLRTEDTATYYC TRDGSGYPSLDFWGQGTQVTVSSGGGGSDIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAP KLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIKGGGGSGGGG SGGGGSEVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIKYADAVKG RFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSGGGGSYELTQPPSASVNVGETV KITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSG YVDSDSKLYVFGSGTQLTVLGPRGGPEQKLISEEDLNSAVDHHHHHH (SEQ ID NO: 749) Group J VH QLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDT STSTATMELRSLRSDDTAVYYCARGRGYSYGIDAFDIWGQGTMVT (SEQ ID NO: 750) VH LSYVLTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQKPGQAPVLVIYKDSERPSGIPERFSGSSSGTTVT LTISGVQAEDEADYYCQSADSSGTYVVFGGGTKLTVLGQP (SEQ ID NO: 751) Group K VH QVQLQQSGPGLVRPSQTLSLSCDISGDSVSSNSATWNWIRQSPSRGLEWLGRTFYRSKWYHDYALSVKSRIT INPDTSKNQFSLQLNSVSPGDTAVYFCVREDIDGRLDYWGQGTLVTVSS (SEQ ID NO: 752) VH QVQLVQSGSELKKPGASVKISCKASGYIFSDNGVNWVRQAPGQGLEWMGWINTKIGNPTYAQGFTGRFVFSL DTSVSTTYLQISSLQAGDTAVYYCAREHDYYYGMDVWGQGTTVTVSS (SEQ ID NO: 753) VH MAEVQLLESGAEVKKPGASVKVSCKASGYTFTSHYMHWVRQAPGQGLEWMGVINPSGGSTSYAQKFQGRVTM TRDTSTSTVYMDLSSLRSEDTAVYYCARRSEAYYHGMDVWGQGTTVTVSS (SEQ ID NO: 754) VH EVQLVQSGSELKKPGASVKISCKASGYIFSDNGVNWVRQAPGQGLEWMGWINTKIGNPTYAQGFTGRFVFSL DTSISTTYLQISSLQAGDTAVYYCAREHDYYYGMDVWGQGTTVTVSS (SEQ ID NO: 755) VH QVQLVESGGGLVQPGGSLRLSCEATGFTFSRYWMHWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISR DNAKNSLYLQMNSLRAEDTAVYYCARDTLEYYGSGILENAMGYYGMDVWGQGTTVTVSS (SEQ ID NO: 756) VH EVQLVESGGGLVRPGGSLRLACAASGFSFSDYYMTWIRQAPGRGLEWIAYISDSGQTVHYADSVKGRFTISR DNTKNSLFLQVNTLRAEDTAVYYCAREDLLGYYLQSWGQGTLVTVSS (SEQ ID NO: 757) VH EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISR DNAKNSLYLQMNSLRAEDTAVYYCAREGEHDAFDIWGQGTMVTVSS (SEQ ID NO: 758) VH QVQLVQSGSELKKPGASVKISCKASGYIFSDNGVNWVRQAPGQGLEWMGWINTKIGNPTYAQGFTGRFVFSL DTSVSTTYLQISSLQAGDTAVYYCAREHDYYYGMDVWGQGTTVTVSS (SEQ ID NO: 759) VH QVQLVQSGSELKKPGASVKISCKASGYIFSDNGVNWVRQAPGQGLEWMGWINTKIGNPTYAQGFTGRFVFSL DTSVSTTYLQISSLQAGDTAVYYCAREHDYYYGMDVWGQGTTVTVSS (SEQ ID NO: 760) VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITR DTSASTAYMELSSLRSEDTAVYYCAKVSAGTESWFDPWGQGTLVTVSS (SEQ ID NO: 761) VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTT DTSTSTAYMELRSLRSDDTAVYYCARGLYGDEDYWGQGTLVTVSS (SEQ ID NO: 762) VH QVQLVQSGSELKKPGASVKISCKASGYIFSDNGVNWVRQAPGQGLEWMGWINTKIGNPTYAQGFTGRFVFSL DTSISTTYLQISSLQAGDTAVYYCAREHDYYYGMDVWGQGTTVTVSS (SEQ ID NO: 763) VH QMQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA DKSISTAYLQWSSLKASDTAMYYCASGVTRKRYSSSWPPFDYWGQGTLVTVSS (SEQ ID NO: 764) VH QVQLQQWGAGLLKSSETLSLSCAVYGGTFRDDHWSWIRQPPGKGLEWIGESHHTGRTIYNPSLRSRVTMSID TSKNEFSLILRSVTAADTATYFCARGNNYVWGNQEDFWGQGTLVTVSS (SEQ ID NO: 765) VH QVQLQQSGPGLVRPSQTLSLSCDISGDSVSSNSATWNWIRQSPSRGLEWLGRTFYRSKWYHDYALSVKSRIT INPDTSKNQFSLQLNSVSPGDTAVYFCVREDIDGRLDYWGQGTLVTVSS (SEQ ID NO: 766) VH EVQLVESGGALVQPGGSLRLSCAVSGFTFSDHYMDWVRQAPGKGLEWVARSRNKGNSYTTEYAASVRGRFTI SRDDSKNSLYLQMNSLKTEDTAVYYCVRVGVVPALDGMDVWGQGTTVTVSS (SEQ ID NO: 767) VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISR DNAKNSLYLQMNSLRAEDTAVYYCARDTLEYYGSGILENAMGYYGMDVWGQGTTVTVSS (SEQ ID NO: 768) VH EVQLLESGGGVVQTGRSLRLSCSDSGSTERSQAMHWVRQTPGKGLEWLAVTSHDGSKTYYADSVKGRFTISR DNSKNTLYLQMNSLRGEDTAVYYCARGGRGYTYDHSFFDYWGQGTLVTVSS (SEQ ID NO: 769) VH QVQLVQSGSELKKPGASVKISCKASGYIFSDNGVNWVRQAPGQGLEWMGWINTKDGNPTYAQGFTGRFVFSL DTSISTTYLQISSLQAGDTAVYYCAREHDYYYGMDVWGQGTTVTVSS (SEQ ID NO: 770) VH QVQLVQSGSELKKPGASVKISCKASGYIFSDNGVNWVRQAPGQGLEWMGWINTKTGNPTYAQGFTGRFVFSL DTSISTTYLQISSLQAGDTAVYYCAREHDYYYGMDVWGQGTTVTVSS (SEQ ID NO: 771) VH QVQLVQSGSELKKPGASVKISCKASGYTFSDNGVNWVRQAPGQGLEWMGWINTKDGNPTYAQGFTGRFVFSL DTSISTTYLQISSLQAGDTAVYYCAREHDYYYGMDVWGQGTTVTVSS (SEQ ID NO: 772) VH QVQLVQSGSELKKPGASVKISCKASGYTFSDNGVNWVRQAPGQGLEWMGWINTKTGNPTYAQGFTGRFVFSL DTSISTTYLQISSLQAGDTAVYYCAREHDYYYGMDVWGQGTTVTVS (SEQ ID NO: 773) VH QVQLVQSGSELKKPGASVKISCKASGYKFSDNGVNWVRQAPGQGLEWMGWINTKSGNPTYAQGFTGRFVFSL DTSISTTYLQISSLQAGDTAVYYCAREHDYYYGMDVWGQGTTVTVSS (SEQ ID NO: 774) VH QVQLVQSGSELKKPGASVKISCKASGYTFSDNGVNWVRQAPGQGLEWMGWINTKSGNPTYAQGFTGRFVFSL DTSISTTYLQISSLQAGDTAVYYCAREHDYYYGMDVWGQGTTVTVSS (SEQ ID NO: 775) VH QVQLVQSGSELKKPGASVKISCKASGYKFSDNGVNWVRQAPGQGLEWMGWINTKDGNPTYAQGFTGRFVFSL DTSISTTYLQISSLQAGDTAVYYCAREHDYYYGMDVWGQGTTVTVSS (SEQ ID NO: 776) VL LPVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSKRPSGVPDRFSGSKSGNT ASLTVSGLQAEDEADYYCSAWDDSLNADVFGGGTKLTVL (SEQ ID NO: 777) VL QPVLTQPPSASGTPGQRVTISCSGSSSNIGTNTVNWYQQVPGTAPKLLIHGNDQRPSGVPDRFSGSKSDTSA SLAITGLQSDDDADYYCSAWDDSLNADVFGGGTKLTVL (SEQ ID NO: 778) VL QAVLTQPPSASATPGQRVTISCSGSDSNIGTNYVYWYQQFPGTAPQPLIYRDNQRPSGVPDRFSGSKSGTSA SLAISGLRSEDEATYFCSTWDDSLNGWVFGGGTKLTVL (SEQ ID NO: 779) VL QPVLTQPRSVSGSPGQSITTSCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNT ASLTISGLQAEDEADYYCSSYTSSSTLEVFGTGTKVTVL (SEQ ID NO: 780) VL YELMQPPSVSGAPGQRVTISCTGSSSNIGAAYDVHWYQQLPGKAPKLVMFANSNRPSGVPDRFSGSKSGTSA SLAITGLQAEDEADYYCQSYDISLRAYVFGTGTKLTVL (SEQ ID NO: 781) VL SYELMQPPSASGTPGQRVTISCSGSSSNIGTNTVNWYQHLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSA SLAISGLQSEDEADYYCATWDDSPNGWVFGGGTKLTVL (SEQ ID NO: 782) VL QAVLTQPPSVSAAPGQRVTISCSGSNSNIADTYVSWYQQLPGTAPRLLIYDNDQRPSGIPDRFSGSKSGTSA TLGITGLQTGDEADYYCGTWDSSLSGVFGTGTKVTVL (SEQ ID NO: 783) VL QSVLTQPASVSGSPGQSVTISCTGSSSDVGAYNFVSWYRQYPGKAPKLLIYEVNKRPSDVPDRFSGSKFGNT ASLTVSGLQADDEADYYCSSYAGSTDVFGTGTKVTVL (SEQ ID NO: 784) VL LPVLTQPPSVSGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYTNNQRPSGVPDRFSGSKSGTSA SLAISGLQSEDEADYYCAAWDESLNGDVFGTGTKVTVL (SEQ ID NO: 785) VL AIRMTQSPSFLSASVGDRVTITCRTSQNIYNYLNWYQQKPGKAPELLIFVASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYFCLQDHSYPYTFGQGTKVEIK (SEQ ID NO: 786) VL LPVLTQPPSVSEVPGQRVTISCSGGISNIGSNAVNWYQHFPGKAPKLLIYYNDLLPSGVSDRFSASKSGTSA SLAISGLRSEDEADYYCAAWDDNLSAYVFATGTKVTVL (SEQ ID NO: 787) VL QSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQHHPGKAPKLMIYEVSKRPSGVPDRFSGSKSAIT ASLTISGLLTEDEADYYCSAWDDSLNADVFGGGTKVTVL (SEQ ID NO: 788) VL DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPKVLITDASSLETGVPSRFSGSGSGTDFT FTISSLQPEDTATYFCQQYDDLPPTFGQGTKLEIK (SEQ ID NO: 789) VL QAGLTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVTKRPSGVSNRFSGSKSGNT ASLTISGLQAEDEADYYCSSYTSSSTYVFGTGTKVTVL (SEQ ID NO: 790) VL QPVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNT ASLAITGLQSDDDADYYCSAWDDSLNADVFGGGTKLTVL (SEQ ID NO: 791) VL QAGLTQPPSVSKGLRQTATLTCTGNSNNIGDQGAAWLQQHQGHPPRLLSYRNNNRPSGISERLSASRSGNIA SLTITGLQPEDEADYYCSAWDSSLSVWVFGGGTKLTVL (SEQ ID NO: 792) VL QAVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSA SLAISGLQSEDEADYYCAAWDDSLNGYVFGTGTKLTVL (SEQ ID NO: 793) VL AIRMTQSPSTLSASVGDRVTITCRASENIRNLLAWYQQKPGKAPELLIHGASTLGTGVPSRFSGGGSGTEFT LTISSLQPDDFATYYCQQYESYFNTFGQGTKVEIK (SEQ ID NO: 794) Group L VH QVQLVESGPGVKKPGSSLKLSCTVSGFTFSSYDYYMHWVRQAPGNGLEWMAVIWYSGSNTYYNDSLKSRFSI TRDNSKNTAYMQLNSLRAEDTAVYYCARAYFGVDVWGQGTLVTVSS (SEQ ID NO: 795) VL DIVMTQSPASLSVSVGDRATISCRASQGIGNTLAWYQQKPGQAPKRLLIYRASQGIGNTLAGVPARFSGDGD GTDFTLTIDDLEEPEDFATYYCQQYDHVPLTFGQGTKLEIK (SEQ ID NO: 796) Group M VH EVQLVQSGAEVKKPGASVKVSCKAFGYTFTTYPIEWMRQAHGQGLEWIGNFHPYNDDTKYNEKFKGRVTMTV DKSTTTVYMELSSLRSEDTAVYYCARENYGSHGGFVYWGQGTLVTVS (SEQ ID NO: 797) VH EVQLVQSGAEVKKPGASVKVSCKAFGYTFTTYPIEWMRQAHGQGLEWIGNFHPYNDDTKYNEKFKGRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARENYGSHGGFVYWGQGTLVTVS (SEQ ID NO: 798) VH EVQLVQSGAEVKKPGASVKVSCKAFGYTFTTYPIEWVRQAPGQGLEWMGNFHPYNDDTKYNEKFKGRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARENYGSHGGFVYWGQGTLVTVS (SEQ ID NO: 799) VH EVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYPIEWVRQAPGQGLEWMGNFHPYNDDTKYNEKFKGRVTITA DKSTSTAYMELSSLRSEDTAVYYCARENYGSHGGFVYWGQGTLVTVS (SEQ ID NO: 800) VH EVQLVQSGAEVKKPGSSVKVSCKAFGYTFTTYPIEWMRQAHGQGLEWIGNFHPYNDDTKYNEKFKGRVTITV DKSTTTVYMELSSLRSEDTAVYYCARENYGSHGGFVYWGQGTLVTVS (SEQ ID NO: 801) VH EVQLVQSGAEVKKPGSSVKVSCKAFGYTFTTYPIEWMRQAHGQGLEWIGNFHPYNDDTKYNEKFKGRVTITA DKSTSTAYMELSSLRSEDTAVYYCARENYGSHGGFVYWGQGTLVTVS (SEQ ID NO: 802) VL ENVLTQSPFSLSASVGDRVTITCRASSSVISSYLHWYQQKPAKAPKLWIYSTSNLASGVPDRFSGSGSGTSY TLTISSLQPEDFATYYCQQYNGYPLTFGGGTKVEIK (SEQ ID NO: 803) VL ENVLTQSPFSLSASVGDRVTITCRASSSVISSYLHWYQQKPAKAPKLFIYSTSNLASGVPSRFSGSGSGTDY TLTISSLQPEDFATYYCQQYNGYPLTFGGGTKVEIK (SEQ ID NO: 804) VL ENVLTQSPFSLSASVGDRVTITCRASSSVISSYLHWYQQKPAKAPKLWIYSTSNLASGVPDRFSGSGSGTSY TLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK (SEQ ID NO: 805) VL ENVMTQSPFSLSASVGDRVTITCRASSSVISSYLHWYQQKPAKAPKLFIYSTSNLASGVPSRFSGSGSGTDY TLTISSLQPEDFATYYCQQYNGYPLTFGGGTKVEIK (SEQ ID NO: 806) VL ENVMTQSPFSLSASVGDRVTITCRASSSVISSYLHWYQQKPAKAPKLFIYSTSNLASGVPSRFSGSGSGTDY TLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK (SEQ ID NO: 807) VL ENVLTQSPFSLSASVGDRVTITCRASSSVISSYLHWYQQKPAKAPKLFIYSTSNLASGVPSRFSGSGSGTDY TLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK (SEQ ID NO: 808) VL ENVLTQSPGTLSLSPGERATLSCRASSSVISSYLHWYQQKPGQAPRLWIYSTSNLASGVPDRFSGSGSGTSY TLTISRLEPEDFATYYCQQYNGYPLTFGGGTKVEIK (SEQ ID NO: 809) VL ENVLTQSPGTLSLSPGERATLSCRASSSVISSYLHWYQQKPGQAPRLWIYSTSNLASGVPDRFSGSGSGTSY TLTISRLEPEDFATYYCQQYNSYPLTFGGGTKVEIK (SEQ ID NO: 810) Group N VH QVQLQQPGAELVKPGASVKLSCKASGYTFTSYYLYWMKQRPGQGLEWIGGVNPSNGGTNFSEKFKSKATLTV DKSSSTAYMQLSSLTSEDSAVYYCTRRDSNYDGGFDYWGQGTTLTVSSAK (SEQ ID NO: 631) VL DIVLTQSPTSLAVSLGQRATISCRASKSVSTSGFSYLHWYQQKPGQPPKLLIFLASNLESGVPARFSGSGSG TDFTLNIHPVEEEDAATYYCQHSWELPLTFGAGTKLELK (SEQ ID NO: 635) Group O VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGSYTIYSADSVKGRFTISR DNAKNTLYLQMSSLRAEDTAVYYCARRGYGSFYEYYFDYWGQGTTVTVS (SEQ ID NO: 1521) VL QIVLTQSPATLSLSPGERATLSCSASSSVSYMYWYQQKPGQAPRLLIYLTSNRATGIPARFSGSGSGTDYTL TISSLEPEDFAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 1522) Group P VH EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWVRQATGQGLEWMGNIYPGTGGSNFDEKFKNRVTITA DKSTSTAYMELSSLRSEDTAVYYCTRWTTGTGAYWGQGTTVTVSS (SEQ ID NO: 1523) VH EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWIRQSPSRGLEWLGNIYPGTGGSNFDEKFKNRFTISR DNSKNTLYLQMNSLRAEDTAVYYCTRWTTGTGAYWGQGTTVTVSS (SEQ ID NO: 1524) VH EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWVRQAPGQGLEWMGNIYPGTGGSNFDEKFKNRFTISR DNSKNTLYLQMNSLRAEDTAVYYCTRWTTGTGAYWGQGTTVTVSS (SEQ ID NO: 1525) VL EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSG SGTEFTLTISSLQPDDFATYYCQNDYSYPYTFGQGTKVEIK (SEQ ID NO: 1526) VL DIQMTQSPSSLSASVGDRVTITCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGIPPRFSGSG YGTDFTLTINNIESEDAAYYFCQNDYSYPYTFGQGTKVEIK (SEQ ID NO: 1527) VL EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSG SGTDFTFTISSLQPEDIATYYCQNDYSYPYTFGQGTKVEIK (SEQ ID NO: 1528) VL DIVMTQTPLSLPVTPGEPASISCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSG SGTDFTFTISSLEAEDAATYYCQNDYSYPYTFGQGTKVEIK (SEQ ID NO: 1529) VL EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSG SGTDFTFTISSLEAEDAATYYCQNDYSYPYTFGQGTKVEIK (SEQ ID NO: 1530) VL EIVLTQSPDFQSVTPKEKVTITCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSG SGTDFTFTISSLEAEDAATYYCQNDYSYPYTFGQGTKVEIK (SEQ ID NO: 1531) VL EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSG SGTDFTFTISSLEAEDAATYYCQNDYSYPYTFGQGTKVEIK (SEQ ID NO: 1532) VL DIQMTQSPSSLSASVGDRVTITCKSSQSLLDSGNQKNFLTWYLQKPGQSPQLLIYWASTRESGVPSRFSGSG SGTDFTFTISSLEAEDAATYYCQNDYSYPYTFGQGTKVEIK (SEQ ID NO: 1533) VL DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSG SGTDFTFTISSLEAEDAATYYCQNDYSYPYTFGQGTKVEIK (SEQ ID NO: 1534) HC EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWVRQATGQGLEWMGNIYPGTGGSNFDEKFKNRVTITA DKSTSTAYMELSSLRSEDTAVYYCTRWTTGTGAYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK (SEQ ID NO: 1535) HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWIRQSPSRGLEWLGNIYPGTGGSNFDEKFKNRFTISR DNSKNTLYLQMNSLRAEDTAVYYCTRWTTGTGAYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK (SEQ ID NO: 1536) HC EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWIRQSPSRGLEWLGNIYPGTGGSNFDEKFKNRFTISR DNSKNTLYLQMNSLRAEDTAVYYCTRWTTGTGAYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK (SEQ ID NO: 1537) HC EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWVRQAPGQGLEWMGNIYPGTGGSNFDEKFKNRFTISR DNSKNTLYLQMNSLRAEDTAVYYCTRWTTGTGAYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK (SEQ ID NO: 1538) HC EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWVRQATGQGLEWMGNIYPGTGGSNFDEKFKNRVTITA DKSTSTAYMELSSLRSEDTAVYYCTRWTTGTGAYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLG (SEQ ID NO: 1539) HC EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWIRQSPSRGLEWLGNIYPGTGGSNFDEKFKNRFTISR DNSKNTLYLQMNSLRAEDTAVYYCTRWTTGTGAYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLG (SEQ ID NO: 1540) LC EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSG SGTEFTLTISSLQPDDFATYYCQNDYSYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 1541) LC DIQMTQSPSSLSASVGDRVTITCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGIPPRFSGSG YGTDFTLTINNIESEDAAYYFCQNDYSYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 1542) LC EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSG SGTDFTFTISSLQPEDIATYYCQNDYSYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 1543) LC DIVMTQTPLSLPVTPGEPASISCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSG SGTDFTFTISSLEAEDAATYYCQNDYSYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 1544) LC EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSG SGTDFTFTISSLEAEDAATYYCQNDYSYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 1545) LC EIVLTQSPDFQSVTPKEKVTITCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSG SGTDFTFTISSLEAEDAATYYCQNDYSYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 1546) LC DIQMTQSPSSLSASVGDRVTITCKSSQSLLDSGNQKNFLTWYLQKPGQSPQLLIYWASTRESGVPSRFSGSG SGTDFTFTISSLEAEDAATYYCQNDYSYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 1547) LC DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSG SGTDFTFTISSLEAEDAATYYCQNDYSYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 1548) LC EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSG SGTDFTFTISSLEAEDAATYYCQNDYSYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 1834) Group Q VH EVQLLESGGGLVQPGGSLRLSCSASGFTFSSYTMNWVRQAPGKGLEWVSGISDTGGNTYYTDSVKGRFTVSR DNSKNTLSLQMNSLRAEDTAVYYCAKDQGGSYPYYFHYWGQGSLVTVSS (SEQ ID NO: 1549) VH EVQLVESGGGLVQPGGSLRLSCAASGFTVSNNYMSWVRQAPGKGLEWVSVIYSGGFTYYTDSVKGRFTISRH NSKNTLYLQMNSLRAEDTAVYYCARYYYDTSDYWTFFDYWGQGTLVTVSS (SEQ ID NO: 1550) VH QVQLVESGGGVVQSGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNIYYSDSVKGRFTISR ANSKNTLYLQMNSLRAEDTAVYYCARPGHWNYFFEYWGQGTLVTVSS (SEQ ID NO: 1551) VH EVQLVESGGGLVQPGGSLRLSCGASGFTFRNYDMHWVRQITGKGLEWVSAIGSAGDTYYPDSVKGRFTISRE NAKNSLYLQMNSLRVGDTAVYYCTRDIHCSSTRCYGMDVWGQGTTVTVSS (SEQ ID NO: 1552) VH EVQLVESGGGLVKPGGSLRLSCAASGFKFSNEWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTI SRDDSKNTLYLQMNSLKTEDTAVYYCTTDQDFWSGYYTGADYYGMDVWGQGTMVTVSS (SEQ ID NO: 1553) VH QMQLQQWGAGLLKPSETLSLTCVVYGGSLNGYYWSWIRQSPGKGLEWIGEIDHSGSTNYNPSLKNRVTMSVD TSKIQFSLKLTSVTVADTAVYYCAREGLLPFDYWGQGTLVTVSS (SEQ ID NO: 1554) VH QLQLQESGPDLVKPSDTLSLTCTVSDDSISSTTYYWAWIRQPPGKGLEWIGSMSYNGNNYYNPSLKSRVAIS AGTSQKQFSLKLTSVTAADTAVYHCARHLGYNGNWYPFDFWGQGILVTVSS (SEQ ID NO: 1555) VH EVQVVESGGGLVEPGRSLRLSCKASGFTFDDYAMHWVRQTPGKALEWVSGINWSGNNIGYADSVKGRFTISK DDAKNSLYLQMNSLRPEDTALYYCTKDISITGTLDAFDVWGQGTMVTVSS (SEQ ID NO: 1556) VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAIIWSDGDSEYNLDSVKGRFTISR DNSKNTLYLQMNSLRVEDSAVYYCARDRDLEDIWGQGTMVTSS (SEQ ID NO: 1557) VH EVQLLESGGVLVQPGGSLRLSCAASGFTFSNFGMTWVRQAPGKGLEWVSGISGGGRDTYFADSVKGRFTISR DNSKNTLYLQMNSLKGEDTAVYYCVKWGNIYFDYWGQGTLVTVSS (SEQ ID NO: 1558) VH EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQVPGKGLEWVSGISWNDGKTVYAESVKGRFIISR DNAKNSLYLEMNSLRAEDTALYYCARDWQYLIERYFDYWGQGTLVTVSS (SEQ ID NO: 1559) VH EVQLVESGGGVVRPGGSLRLSCTASGFTFDDYGMSWVRQAPGKGLEWISGIGWTGGRSSYADSVRGRFTISR DNAKNSLYLQMNSLGAEDTALYYCARDRQWLVQWYFDYWGQGTLVTVSS (SEQ ID NO: 1560) VH EVQLVESGGRVVRPGGSLRLSCAASGFTFDDYGMSWVRQLPGKGLEWVAGISWNDGKTVYAESVKGRFIISR DNAKNSLHLEMNSLRAEDTALYYCARDWQYLIDRYFDFWGQGTLVTVSS (SEQ ID NO: 1561) VH EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGIGWSSGSIGYADSVKGRFTISR DNAKNSLYLQMDSLRPEDSALYYCAKAYTFMITLYFDYWGQGTLVTVSS (SEQ ID NO: 1562) VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYDMHWVRQAPGKGLEWVSGSGWNRGSLGYADSVKGRFTISR DNAKKSLYLQMNSVRVEDTALYYCAKGFVVVSAAYFDYWGQGTLVTVSS (SEQ ID NO: 1563) VH QVQLVQSGAEVKRPGSSVKVSCKVSGVTFRNFAIIWVRQAPGQGLEWMGGIIPFFSAANYAQSFQGRVTITP DESTSTAFMELASLRSEDTAVYYCAREGERGHTYGFDYWGQGTLVTVSS (SEQ ID NO: 1564) VH EVQLVESGGGLVQSGRSLRLSCAASGFTFDDYAMHWVRQPPGKGLEWVSGINWNRGRTGYADSVKGRFTISR DNAKNSLYLQMNDLRVEDTALYYCAKAEQWLDEGYFDYWGQGTLVTVSS (SEQ ID NO: 1565) VH EVQLVESGGGLVQRGGSLRLSCAASGFSFSSYAMNWVRQAPGKGLEWVSTISDSGGSTYYADSVKGRFTISR DNSKNTLSLQMNSLRAEDTAVYYCAKDQGGSYPYYFHYWGQGTLVTVSS (SEQ ID NO: 1566) VH EVQLVESGGGLVQPGRSLRLSCAASGFTFEDYAMHWVRQAPGKGLEWVSGIGWSNVKIGYADSVKGRFTISR DNVRNSLYLQMNSLRTEDTAFYYCVKAYTSMLTLYFDYWGQGTLVTVSS (SEQ ID NO: 1567) VH QVQLVQSGAEVKRPGASVKVSCKASGYTFTSFYMYWVRQAPGQGLEWMGIINPSDGSTSNAQKFQGRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARRVAGDIFDIWGQGTMTVSS (SEQ ID NO: 1568) VH QVQLQESGPGLVKPSETLSLTCTVSGGSISSYHWNWIRQSPGKGLEWIGYIYYIGSTDYNPSLESRVTISVD TSKNQFSLKLSSVTAADTAVYYCARVPVGATGASDVWGQGTMVTVSS (SEQ ID NO: 1569) VH EVQLVESGGSVVRPGGSLRLSCVVSGFTFEDYGLSWVRQIPGKGLEWVSGISWTGGNTGYADSVKGRFTISR DNAKNSLYLQMNSLRAEDTALYHCTRDRQWLMQWYFDYWGQGTLVTVSS (SEQ ID NO: 1570) VH QVQLVESGGGVVQPGRSLRLSCSASGFTFSAYAMHWVRQAPGKGLEWVAAISYGGSDKYYADSVKGRFTISR DNSKNTLYLQMNSLRTDDTAVYYCAKSAHWNFFFDYWGQGTLVTVSS (SEQ ID NO: 1571) VH EVQLVESGGGLVQPGRSLRLSCVASGFALHDYAMHWVRQVPGKGLEWVSSISWNSGVIGYADSLKGRFTISR DNAKNSLYLQMNSLRAEDTALYYCAKGSGSYYVSWFDPWGQGTLVTVSS (SEQ ID NO: 1572) VH QLQLQESGPGLVQPSETLSLTCTVSGDSISSTAYHWDWIRQPPGKGLEWIGTITYNGNTYFNPSLKSRVTIS VDTSKNQFSLKLLSMTAAETAVFYCARHLGYNSDFFPFDFWGQGTLVTVSS (SEQ ID NO: 1573) VH EVQLVESGGGLVRPGGSLRLSCAASGFTFSTYAMAWVRQTPGKGLEGVSAIGGSGDSTYYVDSVKGRFTISR DNSKSTLFLQMNSLRAEDTAVYYCVKVRNYDGSFDIWGQGTMVTVSS (SEQ ID NO: 1574) VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLIWYQQKPGTAPKFLIYAASSLQSGVPSRFSGCGSGTDFT LTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 1575) VL EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFT LTISSLQSGDFAVYYCQQYNNWPLTFGGGTKVEIN (SEQ ID NO: 1576) VL EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFT LTISSLQSGDFAVYYCQQYNNWPLTFGGGTKVEIN (SEQ ID NO: 1577) VL DIQMTQSPSSLSASVGDRVTITCRASQSINNYLNWYQQKPGKAPKLLIYTASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQSYSTPPLTFGQGTQLEIK (SEQ ID NO: 1578) VL DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 1579) VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 1580) VL EIVLTQSPGTLSLSPGERVTLSCRASQSVYSNYLAWYQQNPGQAPRLLIYAASNRATGIPDRFSGSGSGTDF TLTISRLEPEDFAVYYCHQYATSPWTFGQGTKVEIK (SEQ ID NO: 1581) VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRTTGIPDRFSGSGSGTDF TLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 1582) VL DIQMTQSPISVSASVGDRVTITCRASQGISNWLAWYQQKPGIAPKLLIYSASSLQSGVPSRFRGSGSGTDFT LTIGSLQPEDFATYYCQQAHSFPLTFGGGTKVEIK (SEQ ID NO: 1583) VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASNLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK (SEQ ID NO: 1584) VL DIQMTQSPSSLSASVGDSITITCRASLSINTFLNWYQQKPGKAPNLLIYAASSLHGGVPSRFSGSGSGTDFT LTIRTLQPEDFATYYCQQSSNTPFTFGPGTVVDFR (SEQ ID NO: 1585) VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDF TLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 1586) VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 1587) HC EVQLLESGGVLVQPGGSLRLSCAASGFTFSNFGMTWVRQAPGKGLEWVSGISGGGRDTYFADSVKGRFTISR DNSKNTLYLQMNSLKGEDTAVYYCVKWGNIYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK (SEQ ID NO: 1588) HC EVQVVESGGGLVEPGRSLRLSCKASGFTFDDYAMHWVRQTPGKALEWVSGISWSGNNIGYADSVKGRFTISK DDAKNSLYLQMNSLRPEDTALYYCTKDISITGTLDAFDVWGQGTMVTVSSASTKGPSVFPLAPCSRSTSEST AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKV DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK (SEQ ID NO: 1589) HC EVQLVESGGGLVRPGGSLRLSCAASGFTFSTYAMAWVRQTPGKGLEGVSAIGGSGDSTYYVDSVKGRFTISR DNSKSTLFLQMNSLRAEDTAVYYCVKVRNYDGSFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKR VESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK (SEQ ID NO: 1590) HC QVQLVQSGAEVKRPGSSVKVSCKVSGVTFRNFAIIWVRQAPGQGLEWMGGIIPFFSAANYAQSFQGRVTITP DESTSTAFMELASLRSEDTAVYYCAREGERGHTYGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK (SEQ ID NO: 1591) LC DIQMTQSPSSLSASVGDSITITCRASLSINTFLNWYQQKPGKAPNLLIYAASSLHGGVPSRFSGSGSGTDFT LTIRTLQPEDFATYYCQQSSNTPFTFGPGTVVDFRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1592) LC DIQMTQSPISVSASVGDRVTITCRASQGISNWLAWYQQKPGIAPKLLIYSASSLQSGVPSRFRGSGSGTDFT LTIGSLQPEDFATYYCQQAHSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1593) LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDF TLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1594) LC DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1595) Group R VH EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWLRQTPEKRLEWVATMSGGGRDIYYPDSMKGRFTISR DNAKNNLYLQMSSLRSEDTALYYCARQYYDDWFAYWGQGTLVTVSA (SEQ ID NO: 1719) VH QVQLKQSGPGLVQPSQNLSVTCTVSGFSLTTYGVHWVRQSPGKGLEWLGVIWSGGSTDYNAAFISRLTISKD NARSQVFFKMNSLQVNDTAMYYCAREKSVYGNYVGAMDYWGQGTSVTVSS (SEQ ID NO: 1720) VH EVKLVESGGGLVKPGGSLKLSCGASGFTFSSYGMSWVRQTPEKRLEWVATISGGGRDIYYPDSVKGRLTISR DNAKNNLYLQMSSLRSEDTALYYCVRQYYDDWFAYWGQGTLVTVSA (SEQ ID NO: 1721) VH DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNQLEWMAYISYSGYTSYNPSLKSRISITR DTSKNQFFLQLNSVTTEDTATYYCARSLDYDYGTMDYWGQGTSVTVSS (SEQ ID NO: 1722) VH EVKLVESGGGLVKPGGSLKLSCAASGFAFRSYDMSWVRQTPEKILEWVATISGGGSYTYYQDSVKGRFTISR DNARNTLYLQMSSLRSEDTALYYCASPYGPYFDYWGQGTTLTVSS (SEQ ID NO: 1723) VH DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNQLEWMAYISYSGYTSYNPSLKSRISITR DTSRNQFFLQLNSVTTEDTATYYCARSLDYDYGTMDYWGQGTSVTVSS (SEQ ID NO: 1724) VH EVKLVESGGGLVKPGGSLKLSCSASGFSFSYYDMSWVRQTPEKGLEWVATISGGGRNTYFIDSVKGRFTISR DNVKNNLYLLMSSLRSEDTALYYCASPYEGAVDFWGQGTSVTVSS (SEQ ID NO: 1725) VH EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWATISGGGRDTYYLDSVKGRFTISRD NAKNNLYLQMSSLRSEDTALYYCVRQYYDDWFAYWGQGTLVSNSA (SEQ ID NO: 1726) VH QVQLQQSGDELVRPGTSVKMSCKAAGYTFTNNWIGWVKQRPGHGLEWIGDFYPGGGYTNYNEKFKGKATLTA DTSSSTAYMQLSSLTSEDSAIYYCARGYGTNYWYFDVWGAGTTVTVSS (SEQ ID NO: 1727) VH QIHLVQSGPELKKPGETVKISCKASGYTFTNFGMNWVKQAPGKGLKWMGWISGYTREPTYAADFKGRFAISL ETSASTAYLQINDLKNEDMATYFCARDVFDYWGQGTTLTVSS (SEQ ID NO: 1728) VH QIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLKWMGWISGYTREPTYAADFKGRFVISL DTSVSTAYLQISSLKAEDTAVYYCARDVFDYWGQGTLVTVSS (SEQ ID NO: 1729) VH QVQLQESGPGLVKPSQTLSLTCTVSGYSISSDYAWNWIRQPPGKGLEWMAYISYSGYTSYNPSLKSRITISR DTSKNQFSLKLSSVTAADTAVYYCARSLDYDYGTMDYWGQGTLVTVSS (SEQ ID NO: 1730) VL DIVLTQTPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFT LNINSVETEDFGMYFCQQSNSWPLTFGAGTKLELKR (SEQ ID NO: 1731) VL SIVMTQTPKFLLVSAGDRVTITCKASQSVSDDVAWYQQKPGQSPKLLIYYAFNRYTGVPDRFTGSGYGTDFT FTISTVQSEDLAVYFCQQDYRSPWTFGGGTKLEIKR (SEQ ID NO: 1732) VL DIVLTQSPATLSVTPGDSVSLSCRASQSISNDLHWYQQKSHESPRLLIKYVSQSISGIPSRFSGSGSGTDFT LSINSVETEDFGMYFCQQSDSWPLTFGAGTKLELKR (SEQ ID NO: 1733) VL QIVLSQSPAILSASPGEKVTMTCRANSSVSSMHWYQQKPGSSPEPWIYAISNLAFGVPTRFSGSGSGTSYSL TISRVEAEDAATYFCQQWSSRPPTFGGGTKLEIKR (SEQ ID NO: 1734) VL DIQMNQSPSSLSASLGDTITITCHASQSINVWLSWYQQKPGNIPKLLIYRASNLHTGVPSRFSGSGSGTGFT LTISSLQPDDIATYYCQQGQSYPWTFGGGTKLEIKR (SEQ ID NO: 1735) VL QIVLSQSPAILSASPGEKVTMTCRANSSVSSMHWYQQKPGSSPEPWIYAISNLAFGVPARFSGSGSGTSYSL TISRVEAEDAATYFCQQWNSRPPTFGGGTKLEIKR (SEQ ID NO: 1736) VL DIVMTQSHKVMSTSVGDRVSITCKASQDVDNAVAWYQQNPGQSPKLLIKWASTRHHGVPDRFTGSGSGTDFT LTISTVQSEDLADFFCQQYSTFPYTFGGGTKLEIKR (SEQ ID NO: 1737) VL DIVLTQTPATLSVTPGDSVSLSCRASQSLSNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFT LSINSVETEDFGMYFCQQSNSWPLTFGAGTKLEMKR (SEQ ID NO: 1738) VL NIVMTQTPKILFISAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYAFTRYIGVPDRFTGSGYGTDFT FTISTVQAEDLAVYFCQQDYSSPYTFGGGTKLEIKR (SEQ ID NO: 1739) VL DIVLTQSPASLAVSLGQRATISCRASESVDNYGYSFMNWFQQKPGQPPKLLIYRASNLESGIPARFSGSGSR TNFTLTINPVEADDVATYFCQQSNADPTFGGGTNLEIKR (SEQ ID NO: 1740) VL DIVLTQSPASLAVSPGQRATITCRASESVDNYGYSFMNWFQQKPGQPPKLLIYRASNLESGVPARFSGSGSR TDFTLTINPVEANDTANYYCQQSNADPTFGQGTKLEIK (SEQ ID NO: 1741) VL EIVLTQSPATLSLSPGERATLSCRANSSVSSMHWYQQKPGQSPEPWIYAISNLAFGVPARFSGSGSGTDYTL TISSLEPEDFAVYYCQQWSSRPPTFGQGTKLEIK (SEQ ID NO: 1742) Group S VH QVQLVQSGSEVKKSGSSVKVSCKTSGGTFSITNYAINWVRQAPGQGLEWMGGILPIFGAAKYAQKFQDRVTI TADESTNTAYLELSSLTSEDTAMYYCARGKRWLQSDLQYWGQGTLVTVSS (SEQ ID NO: 1743) VL QPVLTQPASVSGSPGQSITISCTGSSSDVGSYDLVSWYQQSPGKVPKLLIYEGVKRPSGVSNRFSGSKSGNT ASLTISGLQAEDEADYYCSSYAGTRNFVFGGGTQLTVL (SEQ ID NO: 1744) Group T VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMYWVKQAPGQGLEWIGGINPSNGGTNYNEKFKNKATLTA DKSTSTAYMELSSLRSEDTAVYYCTRRDYRYDMGFDYWGQGTTVTVSS (SEQ ID NO: 1745) VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEWMGGVNPSNGGTNFNEKFKSRVTITA DKSTSTAYMELSSLRSEDTAVYYCARRDYRYDMGFDYWGQGTTVTVSS (SEQ ID NO: 1746) VH EVQLQQSGPELVKPGASVKISCKASGYTFTNYYMYWVKQSHGKSLEWIGGINPSNGGTNYNEKFKNKATLTV DKSSSTAYMELNSLTSEDSAVYYCARRDYRYDMGFDYWGAGTTVTVSS (SEQ ID NO: 1747) VH EVQLQQSGPVLVKPGASVKMSCKASGYTFTSYYMYWVKQSHGKSLEWIGGVNPSNGGTNFNEKFKSKATLTV DKSSSTAYMELNSLTSEDSAVYYCARRDYRYDMGFDYWGQGTTLTVSS (SEQ ID NO: 1748) VL EIVLTQSPATLSLSPGERATISCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSG TDFTLTISSLEPEDFATYYCQHSRELPLTFGTGTKVEIK (SEQ ID NO: 1749) VL QIVLTQSPAIMSASPGEKVTMTCRASKGVSTSGYSYLHWYQQKPGSSPRLLIYLASYLESGVPVRFSGSGSG TSYSLTISRMEAEDAATYYCQHSRELPLTFGTGTRLEIK (SEQ ID NO: 1750) Group U VH QVQLQQSGAELMKPGASVKMSCKTTGYIFSSYWIGWVKQRPGHGLEWIGKIFPGSGSADYNENFKGKATFTV DTSSNTAYMQLSSLTSEDSAVYYCARGYGNYLYFDVWGAGTTVTVSS (SEQ ID NO: 619) VH DVQLQESGPGLVKPSQSLSLTCTVTGHSITSDYAWNWIRQFPGNKLEWMGYISYSGRTSYNPSLTSRISITR DTSKNQFFLQLNSVTTEDTATYYCARGYALDYWGQGTSVTVSS (SEQ ID NO: 1835) VH EVKLVESGGGLVSPGGSLKLSCAASGFTFSTFGMSWVRQTPEKRLEWVATISGGGSDTYYPDSVQGRFIISR YNAKNNLYLQMNSLRPEDTALYYCARQGYDVYSWFAYWGQGTLVTVSA (SEQ ID NO: 1836) VH EVKLVESGGGLVKPGGSLKLSCAASGFTFSTYGMSWVRQTPEKRLQWVATISGGGSNTYYSDSVKGRFTISR DNAKNNLYLQMSSLRSEDTALYYCARQRDSAWFASWGQGTLVTVSA (SEQ ID NO: 1837) VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSTFGMSWVRQAPGKGLEWVSTISGGGSDTYYPDSVQGRFTISR DNAKNSLYLQMNSLRAEDTAVYYCARQGYDVYSWFAYWGQGTLVTVSS (SEQ ID NO: 1838) VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSTFGMSWVRQAPGKGLEWVATISGGGSDTYYPDSVQGRFTISR DNAKNSLYLQMNSLRAEDTAVYYCARQGYDVYSWFAYWGQGTLVTVSS (SEQ ID NO: 1839) VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYGMSWVRQAPGKGLEWVATISGGGSNTYYSDSVKGRFTISR DDSKNTLYLQMNSLKTEDTAVYYCARQRDSAWFASWGQGTLVTVSS (SEQ ID NO: 1840) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFPHYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDVDYGTGSGYWGQGTLVTVSS (SEQ ID NO: 1841) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFTRQGISWVRQAPGQGLEWMGWISAYNGNTKYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDVDYGSGSGYWGQGTLVTVSS (SEQ ID NO: 1842) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFSTFGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDVDYSSGSGYWGQGTLVTVSS (SEQ ID NO: 1843) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFTRYGISWVRQAPGQGLEWMGWVSAHNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDADYGSGSGYWGQGTLVTVSS (SEQ ID NO: 1844) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFPHYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDAEYGSGSGYWGQGTLVTVSS (SEQ ID NO: 1845) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFTWYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDSEYSSGSGYWGQGTLVTVSS (SEQ ID NO: 1846) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFETYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDAEYSLGSGYWGQGTLVTVSS (SEQ ID NO: 1847) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFRQYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDAEYGSGSGYWGQGTLVTVSS (SEQ ID NO: 1848) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFTWYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDSEYRSGSGYWGQGTLVTVSS (SEQ ID NO: 1849) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFRQYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDAEYRSGSGYWGQGTLVTVSS (SEQ ID NO: 1850) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFTRYGISWVRQAPGQGLEWMGWVSAHNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDADYRSGSGYWGQGTLVTVSSS (SEQ ID NO: 1851) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFPHYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDVDYRTGSGYWGQGTLVTVSS (SEQ ID NO: 1852) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFSTFGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDVDYRSGSGYWGQGTLVTVSS (SEQ ID NO: 1853) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFTRQGISWVRQAPGQGLEWMGWISAYNGNTKYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDVDYRSGSGYWGQGTLVTVSS (SEQ ID NO: 1854) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFPHYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDAEYRSGSGYWGQGTLVTVSS (SEQ ID NO: 1855) VH EVQLVQSGAEVKKPGASVKVSCKASGYRFETYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTT DTSTNTAYMELRSLRSDDTAVYYCARDAEYRLGSGYWGQGTLVTVSS (SEQ ID NO: 1869) VL NIVMTQTPKFLLVSAGDRITITCKASQSVSDDVAWYQQKPGQSPKLLISYAFKRYIGVPDRFTGSGYGTDFT FTISTVQAEDLAVYFCQQNYNSPYTFGGGTKLELKR (SEQ ID NO: 1856) VL QIVLSQSPAILSASPGEKVTMTCRTSSSVNYMHWFQQKPGSSPKPWIYATSKLASGVPARFSGSGSGTSYSL TISRVEAEDAATYFCQQWISDPWTFGGGTKLEIK (SEQ ID NO: 1857) VL SYELTQPPSVSVSPGQTARITCSGDALTTQYAYWYQQKPGQAPVMVIYKDTERPSGIPERFSGSSSGTKVTL TISGVQAEDEADYYCQSADNSITYRVFGGGTKVTVL (SEQ ID NO: 1858) VL SYELTQPPSVSVSPGQTARITCSGDALSEQYAYWYQQKPGQAPVMVIYKDTERPSGIPERFSGSSSGTKVTL TISGVQAEDEADYYCQSADNSITYRVFGGGTKVTVL (SEQ ID NO: 1859) VL SYELTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQKPGQAPVMVIYKDTERPSGIPERFSGSSSGTKVTL TISGVQAEDEADYYCQSADNSITYRVFGGGTKVTVL (SEQ ID NO: 737) VL SYELTQPPSVSVSPGQTARITCSGDALPMQYGYWYQQKPGQAPVMVLYKDTERPSGIPERFSGSSSGTKVTL TISGVQAEDEDYYCQSDNSITYRVFGGGTKVTVL (SEQ ID NO: 1860) VL SYELTQPPSVSVSPGQTARITCSGDALPMQYGYWYQQKPGQAPVMVIYKDTERPSGIPERFSGSSSGTKVTL TISGVQAEDEADYYCQSADNSITYRVFGGGTKVTVL (SEQ ID NO: 1861) VL DIILTQSPASLAVSLGQRAAISCRASESVDNSGISFMSWFQQKPGQPPKLLIYTASNQGSGVPARFSGSGSG TEFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEIR (SEQ ID NO: 1862) VL DIVLTQSPASLAVSLGQRATISCRASENVDDYGVSFMNWFQQKPGQPPKLLIYPASNQGSGVPARFSGSGSG TDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEIK (SEQ ID NO: 1863) VL DIQLTQSPSFLSASVGDRVTITCRASESVDNSGISFMSWYQQKPGKAPKLLIYTASNQGSGVPSRFSGSGSG TEFTLTISSLQPEDFATYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 1864) VL EIVLTQSPATLSLSPGERATLSCRASESVDNSGISFMSWYQQKPGQAPRLLIYTASNQGSGIPARFSGSGSG TDFTLTISSLEPEDFAVYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 1865) VL EIVLTQSPATLSLSPGERATLSCRASENVDDYGVSFMNWYQQKPGQAPRLLIYPASNQGSGIPARFSGSGSG TDFTLTISSLEPEDFAVYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 1866) VL EIVLTQSPGTLSLSPGERATLSCRASENVDDYGVSFMNWYQQKPGQAPRLLIYPASNQGSGIPDRFSGSGSG TDFTLTISRLEPEDFAVYYCQQSKEVPWTFGQGTKVEIK (SEQ ID NO: 1867) VL DIVMTQSPDSLAVSLGERATINCRASENVDDYGVSFMNWYQQKPGQPPKLLIYPASNQGSGVPDRFSGSGSG TDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGGTKLEIK (SEQ ID NO: 1868) Group V VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPGSSLTNYNEKFKNRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARLLTGTFAYWGQGTLVTVSS (SEQ ID NO: 1870) VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPGSSLTNYNEKFKNRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARLSTGTFAYWGQGTLVTVSS (SEQ ID NO: 1871) VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPGSSITNYNEKFKNRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARLTTGTFAYWGQGTLVTVSS (SEQ ID NO: 1872) VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIWPGSSLTNYNEKFKNRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARLLTGTFAYWGQGTLVTVSS (SEQ ID NO: 1873) VH DIVMTQSPDSLAVSLGERATINCKSSQSLWDSGNQKNFLTWYQQKPGQPPKLLIYWTSTRESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQNDYFYPLTFGGGTKVEIK (SEQ ID NO: 1874) VL DIVMTQSPDSLAVSLGERATINCKSSQSLWDSGNQKNFLTWYQQKPGQPPKLLIYWTSYRESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQNDYFYPLTFGGGTKVEIK (SEQ ID NO: 1875) VL DIVMTQSPDSLAVSLGERATINCKSSQSLWDSGNQKNFLTWYQQKPGQPPKLLIYWTSYRESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQNDYFYPHTFGGGTKVEIK (SEQ ID NO: 1876) VL DIVMTQSPDSLAVSLGERATINCKSSQSLWDSTNQKNFLTWYQQKPGQPPKLLIYWTSTRESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQNDYFYPLTFGGGTKVEIK (SEQ ID NO: 1877) HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPGSSLTNYNEKFKNRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARLSTGTFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK (SEQ ID NO: 1878) HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPGSSLTNYNEKFKNRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARLSTGTFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLG (SEQ ID NO: 1879) LC DIVMTQSPDSLAVSLGERATINCKSSQSLWDSGNQKNFLTWYQQKPGQPPKLLIYWTSYRESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQNDYFYPHTFGGGTKVEIKRGTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC (SEQ ID NO: 1880) Group W VH EVQLQESGPELVRPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIDPSNSETSLNQKFKDKATLNV DKSTNTAYMQLSSLTSEDSAVYYCARSRGNYAYEMDYWGQGTSVTVSS (SEQ ID NO: 1972) VH EVQLQESGPELVRPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIEPSSSETSLNQKFKDKATLNV DKSSNTAYMQLSSLTSEDSAVYYCARSRGNYAYEMDYWGQGTSVTVSS (SEQ ID NO: 1973) VH EVQLQESGPELVRPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIDPYSSETSLNQKFKDKATLNV DKISNTAYMQLSSLTSEDSAVYFCARSRGNYAYDMDYWGQGTSVTVSS (SEQ ID NO: 1974) VH EVQLQESGPELVRPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIDPSNSETSLNQKFKDKATLNV DKSSKTAYMQLSSLTSEDSAVYYCARSRGNYAYDMDYWGQGTSVTVSS (SEQ ID NO: 1975) VH EVQLQESGAELVMPGASVKMSCKASGYTFTDYWMHWVKQRPGQGLEWIGAIDTSDSYTSYHQNFKGKATLTE DESSSTAYMQLSSLTSEDSAIYYCARRDYGGFGYWGQGTTLTVSS (SEQ ID NO: 1976) VH EVQLQESGPELVKPGASVKIPCKASGYTFTDYNMDWVKKSHGKSLEWIGDIDPNNGGTIYNQKFKGKATLTV DKSSRTAYMELRSLTSEDTAVYYCARWRSSMDYWGQGTSVTVSS (SEQ ID NO: 1977) VH EVQLQESGPELVKPGASVKIPCRASGYIFTDYNMDWVKQSHGKSLEWIGDIDPNNGGTIYNQKFKDKTTLTV DKSSRTAYMELRSLTSEDTAVYYCARWRSSMDYWGQGTSVTVSS (SEQ ID NO: 1978) VH EVQLQESGPELVKPGASVKIPCKASGYTFTDYNMDWVKQNHGKSLEWIGDIDPNNGDTIYNQKFKGKATLTV DKSSRTAYMELRSLTSEDTAVYYCARWRSSMDYWGQGTSVTVSS (SEQ ID NO: 1979) VH EVQLQESGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDIDPNSGGSIYNQKFKGKATLTV DKSSRTVYMELRSLTSEDTAVYYCARWRSSMDYWGQGTSVTVSS (SEQ ID NO: 1980) VH EVQLQESGPELVKPGASVKITCKASGYTFTDYNMDWVKQSHGKSLEWIGDIDPNNGGTIYNQKFKGKATLTV DKSSNTAYMELRSLASEDTAVYYCARWRSSMDYWGQGTSVSVSS (SEQ ID NO: 1981) VH EVQLQESGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDIDPNNGGTIYNQNFKGKATLTV DKSSSTAYMELRSLTSEDTAVYYCARWRSSMDYWGQGTSVTVSS (SEQ ID NO: 1982) VH EVQLQESGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDIDPNNGGIIYNQKFKGKAALTV DKSSSTAYMELRSLTSEDTAVYYCARWRSSMDYWGQGTSVTVSS (SEQ ID NO: 1983) VH EVQLQESGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDIDPNNGGIIYNQKFKGKAALTV DKSSSTAYMELRSLTSEDTAVYYCTRWRSSMDYWGQGTSVTVSS (SEQ ID NO: 1984) VH EVQLQESGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDIDPNNGNTIYNQKFKGKATLTV DKSSSTAYMELRSLTSEDTAVYYCTKWRSSMDYWGQGTSVTVSS (SEQ ID NO: 1985) VH EVQLQESGPELVRPGASVKIPCKASGYTFTDYNMDWVMQSHGKSLEWIGDIDPNNGGTIYNQKFKGKATLTV DKSSSTAYMELRSLTSEDTAVYYCTRWRSSMDYWGQGTSVTVSS (SEQ ID NO: 1986) VH EVQLQESGPELVKPGASVKIPCKASGYTFTDYNVDWVKQSHGKSLEWIGDIDPNNGGTFYNQKFKGKATLTV DKSSSTAHMELRSLTSEDTAVYYCVRWRSSMDYWGQGTSVTVSS (SEQ ID NO: 1987) VH EVQLQESGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDIDPNTGTTFYNQDFKGKATLTV DKSSSTAYMELRSLTSEDTAVYYCARWRSSMDYWGQGTSLTVSS (SEQ ID NO: 1988) VH EVQLQESGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGVIDPGTGGTAYNQKFKVKALLTA DKSSNTAYMELRSLTSEDSAVYYCTSEKFGSNYYFDYWGQGTTLTVSS (SEQ ID NO: 1989) VH EVQLQESGAELVRPGASVTLSCKASGYTFTDYEIHWVKQTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTA DKSSSTAYMELRSLTSEDSAVYYCTSEKFGSSYYFDYWGQGTTFTVSS (SEQ ID NO: 1990) VH EVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGSPTYNPSLKSQFSITR DTSKNQFFLQLNSLTTEDTATYYCARGLGGHYFDYWGQGTTLTVSS (SEQ ID NO: 1991) VH EVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGSPTYNPSLKSQFSITR DTSKNQFFLQLNSVTTEDTATYYCARGLGGHYFDYWGQGTTLTVSS (SEQ ID NO: 1992) VH EVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGFIHYSGDTNYNPSLKSRFSITR DTSKNQFFLHLNSVTPEDTATYYCASPSRLLFDYWGHGTTLTVSS (SEQ ID NO: 1993) VH EVQLQESGPGLVAPSQSLSITCTVSGFSLTNYGVDWVRQSPGKGLEWLGVIWGVGSTNYNSALKSRLSISKD NSKSQVFLKMNSLQTDDTAMYYCASDGFVYWGQGTLVTVSS (SEQ ID NO: 1994) VH EVQLQESGPGLVAPSQSLSITCTVSGFSLTSYGVDWVRQSPGKGLEWLGVIWGIGSTNYNSALKSRLSISKD NSKSQVFLKMNSLQSDDTAMYYCASDGFVYWGQGTLVTVSS (SEQ ID NO: 1995) VH EVQLQESGPSLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLGVIWRGGNTDYNAAFMSRLSITKD NSKSQVFFKMNSLQADDTAIYYCAASMIGGYWGQGTTLTVSS (SEQ ID NO: 1996) VH EVQLQESGPSLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWRGGNTDYNAAFMSRLSITKD NSKSQVFFKFHSLQTDDTAIYYCAASMIGGYWGQGTTLTVSS (SEQ ID NO: 1997) VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGMIDPSNSETSLNQKFQGRVTMTV DKSTNTVYMELSSLRSEDTAVYYCARSRGNYAYEMDYWGQGTLVTVSS (SEQ ID NO: 1998) VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGMIDPSNSETSLNQKFQGRVTLNV DKSTNTAYMELSSLRSEDTAVYYCARSRGNYAYEMDYWGQGTLVTVSS (SEQ ID NO: 1999) VH EVQLVQSGTEVTKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWLGMIDPSNSETTLNQKFQGRVTMTV DKSTNTVYMELTSLRSEDTAVYYCARSRGNYAYEMDYWGQGTLVTVSS (SEQ ID NO: 2000) VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGIIDPGTGGTAYNQKFQGRVTMTA DKSTSTVYMELSSLRSEDTAVYYCTSEKFGSNYYFDYWGQGTLVTVSS (SEQ ID NO: 2001) VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGIIDPGTGGTAYNQKFQGRVTMTA DKSTNTVYMELSSLRSEDTAVYYCTSEKFGSNYYFDYWGQGTLVTVSS (SEQ ID NO: 2002) VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQRLEWMGVIDPGTGGTAYNQKFQGRVTITA DKSASTAYMELSSLRSEDTAVYYCTSEKFGSNYYFDYWGQGTLVTVSS (SEQ ID NO: 2003) VL DIVLTQTPAIMSASPGEKVTLTCSASSSVSSNYLYWYQQRPGSSPKLWIYSTSNLASGVPARFSGSGSGTSY SLTISSMEAEDAASYFCHQWSSYPPTFGSGTKLEIK (SEQ ID NO: 2004) VL DIVITQTTAIMSASPGEKVTLTCSASSSVSSNYLYWYQQRPGSSPKLWIYSTSNLASGVPARFSGSGSGTSY SLTISSMEAEDAASYFCHQWSSYPPTFGSGTKLEIK (SEQ ID NO: 2005) VL DIVMTQTPATMSASPGEKVTLTCSASSSVNSNYLYWYQQKPGSSPKVWIYSTSNLASGVPARFSGSGSGTSY SLTISSMEAEDAASYFCHQWSSYPPTFGSGTKLELK (SEQ ID NO: 2006) VL DIVMTQTTATMSASPGEKVTLTCSASSSVNSNYLYWYQQKPGSSPKVWIYSTSNLASGVPARFSGSGSGTSY SLTISSMEAEDAASYFCHQWSSYPPTFGSGTKLELK (SEQ ID NO: 2007) VL DIVLTQSTAIMSASPGEKVTLTCSASSGVNSNYLYWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSY SLTISSVEAEDAASYFCHQWSSYPPTFGSGTKLEIK (SEQ ID NO: 2008) VL DIVLTQTPSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYS LTISNLEPEDIATYYCQQYSKLPWTFGGGTKLEIK (SEQ ID NO: 2009) VL DIVLTQSPSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYS LTISNLEPEDIATYYCQQYSKLPWTFGGGTKLEIK (SEQ ID NO: 2010) VL DIVITQSPSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYS LTISNLEPEDIATYYCQQYSELPWTFGGGTKLEIK (SEQ ID NO: 2011) VL DIVMTQSPSSLSASLGDRVTISCSASQGISNYLNWYQQRPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYS LTISNLEPEDIATYYCQQYSNLPWTFGGGTKLEIK (SEQ ID NO: 2012) VL DIVMTQSPSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYS LTISNLEPEDIATYYCQQYSELPWTFGGGTKLEIK (SEQ ID NO: 2013) VL DIVMTQSTSSLSASLGDRVTISCSASQGISHYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYS LTIRNLEPEDIATYYCQQYSELPWTFGGGTKLEIK (SEQ ID NO: 2014) VL DIVMTQSPSSLSASLGDRVTISCSASQGISHYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYS LTIRNLEPEDIATYYCQQYSELPWTFGGGTKLEIK (SEQ ID NO: 2015) VL DIVMTQSPSSLSASLGDRVTISCSASQDISSYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYS LTISNLEPEDIATYYCQQYSELPWTFGGGTKLEIK (SEQ ID NO: 2016) VL DIVMTQTPSSLSASLGDRVTISCSASQGISYYLNWYQQKPDGTIKLLIYYTLSLHSGVPSRFSGSGSGTDYS LTISNLEPEDIATYYCQQYSELPWTFGGGTKLEIK (SEQ ID NO: 2017) VL DIVMTQTPSSMSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYS LTISNLEPEDIATYYCQQYSYLPWTFGGGTKLEIK (SEQ ID NO: 2018) VL DIVMTQTPSSLSASLGDRVTISCSASQGIGNYLNWYQQKPDGTVKLLIYYTSNLHSGVPSRFSGRGSGTDYS LTISNLEPEDIATYYCQQYSNLPWTFGGGTKLEIK (SEQ ID NO: 2019) VL DIVMTQSPSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSNLHSGVPSRFSGSGSGTDYS LTISDLAPEDIATYYCQQYSYLPWTFGGGTKLEIK (SEQ ID NO: 2020) VL DIVITQSPLSLPVGLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIK (SEQ ID NO: 2021) VL DIVLTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTNFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK (SEQ ID NO: 2022) VL DIVLTQSPLSLPVSLGDQASISCRSSQSIVHSDGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDLGIYYCFQGSHVPLTFGAGTKLELK (SEQ ID NO: 2023) VL DIVITQTPLSLPVSLGDQASISCRSSQTIVHSDGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLEIK (SEQ ID NO: 2024) VL DIVMTQSTLSLPVSLGDQVSISCRSSQSIVHSDGNTYLEWYLQKPGQSPNLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK (SEQ ID NO: 2025) VL DIVLTQDELSNPVTSGESVSISCRSSKSLLYKDGKTYLNWFLQRPGQSPQVLIYFMSTRASGVSDRFSGSGS GTDFTLEISRVKAEDVGVYYCQQLVDFPFTFGSGTKLELK (SEQ ID NO: 2026) VL DIVMTQDELYNPVTSGESVSISCRSSKSLLYKDGKTYLNWFLQRPGQSPQVLIYFMSTRASGVSDRFSGSGS GTDFTLEISRVKAEDVGVYYCQQLVDFPFTFGSGTKLEIK (SEQ ID NO: 2027) VL DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGTQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSG SGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLEIK (SEQ ID NO: 2028) VL DIVLTQTTATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFT LTVNSVEPEDVGVYYCQNGHSYPYTFGGGTKLEIK (SEQ ID NO: 2029) VL DIVLTQSPDTLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFT LSINSVEPEDVGVYYCQNGHSYPYTFGGGTKLELK (SEQ ID NO: 2030) VL EIVLTQSPATLSLSPGERATLSCRASSSVSSNYLYWYQQKPGQAPRLLIYSTSNRATGIPARFSGSGSGTDY TLTISSLEPEDFAVYYCHQWSSYPPTFGQGTKLEIK (SEQ ID NO: 2031) VL DIVLTQSPATLSLSPGERATLSCRASSSVSSNYLYWYQQKPGQAPRLLIYSTSNLATGIPARFSGSGSGTDY TLTISSLEPEDFAVYFCHQWSSYPPTFGQGTKLEIK (SEQ ID NO: 2032) VL DIVLTQSPGTLSLSPGEKVTLSCRASSSVSSNYLYWYQQKPGQAPRLVIYSTSNLATGIPDRFSGSGSGTDY TLTISRLEPEDFAVYFCHQWSSYPPTFGQGTKVEIK (SEQ ID NO: 2034) VL DVVMTQSPLSLPVTLGQPASISCRSSQTIVHSDGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIK (SEQ ID NO: 2035) VL DIVMTQSPLSLPVTLGQPASISCRSSQTIVHSDGNTYLEWYQQRPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIK (SEQ ID NO: 2036) VL DIVMTQTPLSSPVTLGQPASISCRSSQTIVHSDGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGA GTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIK (SEQ ID NO: 2037)

In some embodiments, the activatable anti-PD-1 antibody includes a CDR sequence shown in Table 8, a combination of VL CDR sequences (VL CDR1, VL CDR2, VL CDR3) selected from the group consisting of those combinations shown in a single row Table 8, a combination of VH CDR sequences (VH CDR1, VH CDR2, VH CDR3) selected from the group consisting of those combinations shown in Table 8, or a combination of VL CDR and VH CDR sequences (VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, VH CDR3) selected the group consisting of those combinations shown in Table 8. The CDR regions were defined according to AbM definition as described in the CDR definition table in Andrew C. R. Martin's Bioinformatics Group website at UCL.

TABLE 8 CDR Sequences for Antibodies and Activatable Antibodies that Bind PD-1 VH VL CDR1 CDR2 CDR2 CDR3 AB (SEQ ID (SEQ ID CDR3 (SEQ CDR1 (SEQ ID (SEQ ID (SEQ ID Name NO) NO) ID NO) NO) NO) NO) M13 GFTFSGYAMS YISNSGGNAH EDYGTSPFVY RASESVDNYGIS AASNQGS QQSKDVPWT (653) (658) (664) FMN (669) (678) (683) M19 GYTFTDYYMD YIYPKNGGSS KVVATDY KSSQSLLYSSNQ WASIRES QQCDSYPWT (654) (659) (665) KNYL (670) (679) (684) M3 GFTFSNYAMS YISNGGGDTH ENYGTSPFVY RASESVDNYGIS AASNQGS QQSKDVPWT (655) (660) (666) FMN (669) (678) (683) M5 GFSFSSYDMS TISGGGRYTY NYYGFDY KASQDVGTAVA WASTRHT QQYSSYPWT (656) (661) (667) (671) (680) (685) M14 GFTFSSYGMS TISGGGRDIY LYLGFDY LASQTIGTWLA AATSLAD QQLYSIPWT (657) (662) (668) (672) (681) (686) A GFTFSGYAMS YISNSGGNAH EDYGTSPFVY (653) (658) (664) Ab GFTFSGYAMS YISNSGGNAH EDYGTSPFVY (653) (658) (664) Ae GFTFSGYAMS YISNSGGNTH EDYGTSPFVY (653) (663) (664) Af GFTFSGYAMS YISNSGGNTH EDYGTSPFVY (653) (663) (664) Ba GYTFTDYYMD YIYPKNGGSS KVVATDY (654) (659) (665) Bb GYTFTDYYMD YIYPKNGGSS KVVATDY (654) (659) (665) C GFTFSNYAMS YISNGGGDTH ENYGTSPFVY (655) (660) (666) Ca GFTFSNYAMS AYISNQGGDT ENYGTSPFVY (655) H (2041) (666) D GFSFSSYDMS TISGGGRYTY NYYGFDY (656) (661) (667) 1.0 RASESVDNYGIS AASNQGS QQSKDVPWT FMN (673) (678) (683) 1.1 RASESVDNYGIS AASNQGS QQSKDVPWT FMN (673) (678) (683) 1.2 RASESVDQYGIS AASNQGS QQSKDVPWT FMN (674) (678) (683) 1.4 RASESVDSYGIS AASNQGS QQSKDVPWT FMN (675) (678) (683) 1.5 RASESVDAYGIS AASNQGS QQSKDVPWT FMN (676) (678) (683) 1.6 RASESVDNYGIS AASDQGS QQSKDVPWT FMN (673) (682) (683) cl RASESVDAYGIS AASNQGS QQSKDVPWT 1.7 FMN (676) (678) (683) 1.9 RASESVDAY AASNQGS QQSKDVPWT (676)GISFMN (678) (683) 1.10 RASESVDAYGIS AASNQGS QQSKDVPWT FMN (676) (678) (683) 2 KSSQSLLYSSNQ WASIRES QQSDSYPWT KNYLA (677) (679) (687) 4 KASQDVGTAVA WASTRHT QQYSSYPWT (671) (680) (685)

In some embodiments, the activatable anti-PD-1 antibody comprises or is derived from an antibody that is manufactured, secreted or otherwise produced by a hybridoma, such as, for example, the hybridoma(s) disclosed in U.S. Pat. No. 8,927,697 and US Patent Application Publication Nos. US 2011-0171215 and US 2015-0152180, and deposited at the European Collection of Cell Cultures (ECACC) under Accession Number 08090902; 08090903; and Ser. No. 08/090,901.

In some embodiments, the activatable anti-PD-1 antibody comprises or is derived from an antibody that is manufactured, secreted or otherwise produced by a hybridoma, such as, for example, the hybridoma(s) disclosed in US Patent Application Publication No. US 2014-0335093, and deposited at the Collection Nationale De Cultures De Microorganismes (CNCM) under deposit number 1-4122.

In some embodiments, the activatable anti-PD-1 antibody comprises or is derived from an antibody that is manufactured, secreted or otherwise produced by a hybridoma, such as, for example, the hybridoma(s) disclosed in PCT Publication Nos. WO 2015/058573 or WO 2014/206107, and deposited at the China Culture Collection Committee General Microbiology Center under Accession no. 8351.

In some embodiments, the activatable anti-PD-1 antibody comprises or is derived from an antibody that is manufactured, secreted or otherwise produced by a hybridoma, such as, for example, the hybridoma(s) disclosed in US Patent Application Publication No. US2011177088, and deposited at the Collection Nationale De Cultures De Microorganismes (CNCM) under deposit number 1-3745.

In some embodiments, the activatable anti-PD-1 antibody includes a CDR sequence shown in Table 9, a combination of VL CDR sequences selected from the group consisting of those combinations shown in Table 9, and/or a combination of VH CDR sequences selected from the group consisting of those combinations shown in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group A in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group A in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group A in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group A in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group B in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group B in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group B in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group B in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group C in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group C in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group C in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group C in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group E in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group E in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group E in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group E in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group F in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group F in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group F in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group F in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group G in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group Gin Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group Gin Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group G in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group H in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group H in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group H in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group H in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group L in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group L in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group L in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group L in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group M in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group M in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group M in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group M in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group N in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group N in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group N in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group N in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group O in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group O in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group O in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group O in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group P in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group P in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group P in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group P in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group Q in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group Q in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group Q in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group Q in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group R in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group R in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group R in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group R in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group T in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group T in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group T in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group T in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group U in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group U in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group U in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group U in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group V in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group V in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group V in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group V in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group W in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group W in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group W in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group W in Table 9.

In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group X in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group X in Table 9. In some embodiments, the activatable anti-PD-1 antibody includes a combination of heavy chain CDR sequences selected from the group consisting of the combinations shown in Group X in Table 9, and a combination of light chain CDR sequences selected from the group consisting of the combinations shown in Group X in Table 9.

TABLE 9 Additional CDR Sequences for Antibodies and Activatable Antibodies that Bind PD-1 VH VL CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) Group A NYGMH VIWYDGSNKYYADSVKG NDDY (700) RASQSVSSYLA DASNRAT QQRSNWPLT (688) (694) (704) (706) (710) NYGFH VIWYDGSKKYYADSVKG GDDY (701) RASQSVSSYLA DTSNRAT QQRSNWPLT (689) (695) (704) (707) (710) NYGMH LIWYDGSNKYYADSVKG NVDH (702) RASQSVSSYLA DASNRAT QQSSNWPRT (688) (696) (704) (706) (711) NSGMH VIWYDGSKRYYADSVKG NDDY (700) RASQSVSSYLA DASNRAT QQSSNWPRT (690) (697) (704) (706) (711) RSSFFWG SIYYSGSTYYNPSLKS DYDILTGDEDY RASQGISSWLA AASNLRS QQYYSYPRT (691) (698) (703) (705) (708) (712) SYGFH VIWYDGSKKYYADSVKG GDDY (701) RASQSVSSYLA DASNRAT QQRSNWPLT (692) (695) (704) (706) (710) RSSYFWG SIFYSGETYFNPSLKS DYDILTGDEDY RASQGISSWLA AASSLQS QQYYSYPRT (693) (699) (703) (705) (709) (712) Group B SYYLY GVNPSNGGTNFSEKFK RDSNYDGGFDY RASKSVSTSGFSY LASNLES QHSWELPLT (713) S (715) (717) LH (719) (721) (723) NYYMY GINPSNGGTNFNEKFK RDYRFDMGFDY RASKGVSTSGYSY LASYLES QHSRDLPLT (714) N (716) (718) LH (720) (722) (724) AASGFTFS TITGGGRNTYYPDSVK QGYDGYTWFAY RASESVDNSGISF AASNPGS QQSKEVPWT SYAMS G (817) (823) MN (830) (836) (840) (627) AASGFTFS TISGGGSNTYYPDSVK IYDVAWFAY RSSQTIVHSDGNT AASNQGS FQGSHVPYT SFGMS (818) (824) YLE (831) (678) (841) (811) KGSGYSFT VISTHYGDTVYNQRFK EGYGSLFYFDQ RASESVDSYGNSF KVSNRFS QQNNEVPLT DYALH G (1832) (825) MN (832) (837) (842) (812) KASGYAFT YIDLYNGDTSYNEKF EGRLSFDY RSSQSIVQSNGNT LASNLDS FQGSHVPYT SYNIY (819) (826) YLE (833) (838) (843) (813) AASGFTFS YISGGGGNTYYPDTL ISLTGIFDY RSSQTIVHGNGNT SASTLAS QQGFGTSNVE NYDMS (820) (827) YLE (834) (839) NP (844) (814) AASGFTFN TISGGGSYTYYPDSVQ GNYVYVMDY QASENIYSSLA SYGMS G (821) (828) (835) (815) KGSGYIFT VISTYYSNINYNQKFK EGFGRPYWYFD DYVMH G (822) V (829) (816) Group C GYSITSDY YINYSGSTSYNPSLKS WIGSSAWYFDV RSGQNIVHSNGNTY KVSNRFF FQGSHVPFT AWN (847) (849) LE (851) (853) (855) (845) GYTFTTYY GINPSNGGINFNEKFK RDYRYDRGFDY RASKSVSTSGFNYI LASNLES QHSRELPLT LY (846) (848) (850) H (852) (721) (856) Group E SYGIS WISAYNGNTNYAQ DADYSSGSGY SGDALPKQYAY KDTERPS QSADNSITYR (857) KLQG (861) (865) (869) (872) V (876) SYYIH IINPRGATISYAQ AGIYGFDFDY TGTSNDVGGYNYVS DVTNRPS SSYTIVTNFE (858) KFQG (862) (866) (870) (873) VL (877) SGAYYWS YIYYNGNTYYNPS ASDYVWGGYRYM SGSNSNIGSNSVN GNNQRPS AAWDDSLNGP (859) LRS (863) DAFDI (867) (871) (874) V (878) SSYWMS AISGSGGSTYYAD ENWGSYFDL RASQGISSWLA KASTLES QQSYSTPWT (860) SVKG (864) (868) (705) (875) (879) Group F SSWIH YIYPSTGFTEYNQ WRDSSGYHAMDY RASQSVSTSGYSYMH FGSNLES QHSWEIPYT (880) KFKD (881) (882) (883) (884) (885) Group L GFTFSSYD VIWYSGSNTYYND AYFGVDV RASQGIGNTLA RASQGIG QQYDHVPLT YYMH SLKS (887) (888) (889) NTLA (891) (886) (889) Group G GFSLTSYG VIWAGGSTNYNSA GFSLTSYGVH KASQSVSNDVA YAFHRFT VH (892) LMS (895) (896) (898) (900) VIWAGGST ARAYGNYWYIDV ARAYGNYWYIDV KSSESVSNDVA NYNPSLKS (896) (897) (899) (893) VIYAGGST HQAYSSPYT NYNPSLKS (895) (894) Group H DYEMH VIESETGGTAYNQ EGITTVATTYYW RSSQSIVHSNGNTYLE KVSNRFS FQGSHVPLT (901) KFKG (902) YFDV (903) (904) (905) (906) Group M GYTFTTYP NFHPYNDDTKYNE RASSSVISSYLH STSNLAS QQYNGYPLT IE (907) KFK (908) (909) (910) (911) QQYNSYPLT (912) Group N SYYLY GVNPSNGGTNFSE RDSNYDGGFDY RASKSVSTSGFSYLH LASNLES QHSWELPLT (713) KFK (1596) (717) (719) (721) (723) Group P GYTFTTY YPGTGG (1597) WTTGTGAY SQSLLDSGNQKNF WAS DYSYPY (854) (1598) (1599) (1600) (1601) TYWMH NIYPGTGGSNFDE KSSQSLLDSGNQKNFL WASTRES QNDYSYPYT (1602) KFK (1603) (1833) (1604) (1605) GYTFTTYW MH (1606) Group Q GFTFSSYT ISDTGGNT AKDQGGSYPYYF QSISSY (1610) AAS QQSYSTPPIT (1607) (1608) HY (1609) (1611) (1612) GFTVSNNY IYSGGFT ARYYYDTSDYWT QSVSSN (1616) GAS QQYNNWPLT (1613) (1614) FFDY (1615) (1617) (1618) GFTFSSYG IWYDGSNI ARPGHWNYFFEY QSINNY (1622) TAS QQSYSTPPLT (1619) (1620) (1621) (1623) (1624) GFTFRNYD IGSAGDT TRDIHCSSTRCY QSISNY (1628) AAS QQSYSTPPIT (1625) (1626) GMDV (1627) (1611) (1612) GFKFSNEW IKSKTDGGTT TTDQDFWSGYYT AAS QQSYSTPPIT (1629) (1630) GADYYGMDV (1611) (1612) (1631) GGSLNGYY IDHSGST AREGLLPFDY QSVYSNY (1635) AAS HQYATSPWT (1632) (1633) (1634) (1611) (1636) DDSISSTT MSYNGNN ARHLGYNGNWYP QSVSSSY (1639) GAS QQYGSSPWT YY (622) FDF (1638) (1640) (1641) (1637) GFTFDDYA INWSGNNI TKDISITGTLDA QGISNW (1645) SAS QQAHSFPLT (1642) (1643) FDV (1644) (1646) (1647) GFTFSSYG IWSDGDSE ARDRDLEDI QGIRND (1650) AAS LQHNSYPLT (1619) (1648) (1649) (1611) (1651) GFTFSNFG ISGGGRDT VKWGNIYFDY LSINTF (1655) AAS QQSSNTPFT (1652) (1653) (1654) (1611) (1656) GFTFDDYG ISWNDGKT ARDWQYLIERYF QSVSSSY (1639) GAS QQYGSSPWT (1657) (1658) DY (1659) (1640) (1641) GFTFDDYG IGWTGGRS ARDRQWLVQWYF QSISSY (1610) AAS QQSYSTPPIT (1657) (1660) DY (1661) (1611) (1612) GFTFDDYG ISWNDGKT ARDWQYLIDRYF (1657) (1658) DF (1662) GFTFDDYA IGWSSGSI AKAYTFMITLYF (1642) (1663) DY (1664) GFTFDDYD SGWNRGSL AKGFVVVSAAYF (1665) (1666) DY (1667) GVTFRNFA IIPFFSAA AREGERGHTYGF (1668) (1669) DY (1670) GFTFDDYA INWNRGRT AKAEQWLDEGYF (1642) (1671) DY (1672) GFTFEDYA ISDSGGST AKDQGGSYPYYF (1673) (1674) HY (1609) GYTFTSFY IGWSNVKI VKAYTSMLTLYF (1675) (1676) DY (1677) GGSISSYH INPSDGST ARRVAGDIFDI (1678) (1679) (1680) GFTFEDYG IYYIGST ARVPVGATGASD (1681) (1682) V (1683) GFTFSAYA ISWTGGNT TRDRQWLMQWYF (1684) (1685) DY (1686) GFALHDYA ISYGGSDK AKSAHWNFFFDY (1687) (1688) (1689) GDSISSTA ISWNSGVI AKGSGSYYVSWF YH (1717) DP (1718) (1687) GFTFSTYA ITYNGNT ARHLGYNSDFFP (1690) (1691) FDF (1692) IGGSGDST VKVRNYDGSFDI (1693) (1694) Group R SYGMS TMSGGGRDIYYPD QYYDDWFAY RASQSISNNLH YASQSIS QQSNSWPLT (1751) SMKG (1752) (1753) (1754) (1755) (1756) TYGVH VIWSGGSTDYNAA EKSVYGNYVGAM KASQSVSDDVA YAFNRYT QQDYRSPWT (1757) FIS (1758) DY (1759) (1760) (1761) (1762) SYGMS TISGGGRDIYYPD QYYDDWFAY RASQSISNDLH YVSQSIS QQSDSWPLT (1751) SVKG (1763) (1753) (1764) (1765) (1766) SDYAWN YISYSGYTSYNPS SLDYDYGTMDY RANSSVSSMH AISNLAF QQWSSRPPT (1767) LKS (1768) (1769) (1770) (1771) (1772) SYDMS TISGGGSYTYYQD PYGPYFDY HASQSINVWLS ASNLHT QQGQSYPWT (1773) SVKG (1774) (1775) (1776) (1777) (1778) SDYAWN YISYSGYTSYNPS SLDYDYGTMDY RANSSVSSMH AISNLAF QQWNSRPPT (1767) LKS (1768) (1769) (1770) (1771) (1779) YYDMS TISGGGRNTYFID PYEGAVDF KASQDVDNAVA WASTRHH QQYSTFPYT (1780) SVKG (1781) (1782) (1783) (1784) (1785) SYGMS TISGGGRDTYYLD QYYDDWFAY RASQSLSNNLH YASQSIS QQSNSWPLT (1751) SVKG (1786) (1753) (1787) (1755) (1756) NNWIG DFYPGGGYTNYNE GYGTNYWYFDV KASQSVSNDVA YAFTRYI QQDYSSPYT (1788) KFKG (1789) (1790) (1791) (1792) (1793) NFGMN WISGYTREPTYAA DVFDY (1796) RASESVDNYGYSFMN RASNLES QQSNADPT (1794) DFKG (1795) (1797) (1798) (1799) Group T GYTFTSYY GVNPSNGGTNFNE RDYRYDMGFDY RASKGVSTSGYSYLH LASYLE QHSRELPLT MY KFKS (1801) (1802) (1803) (1804) (1805) (1800) GYTFTNYY GINPSNGGTNYNE RDYRYDMGFDY MY KFKN (890) (1802) (1806) Group U G W (I/V) S A (Q/—) GY S G D A L Q Q (T/R/I) (Y/H) N G N T (G/D)(N/V)Y (P/T/S) (N/S/W) (F/L) (K/N) Y A Q K (L/S)(Y/W) (M/T/E/K) Q Y (Y/K/I) (S/E/ L Q G (1882) (D/A)(Y/V) (G/A) Y (1884) (N/E/S) T/P/R) (1883) (S/V/D/T) (T/S/ P (Y/W) T H/Q/R/ (1885) W) (F/Y/Q) (1881) (T/R/W/Q/ (T/A) I S G D H/S) (S/G) G (A/V/S)(D/E) Y (Y/F/Q) (S/G).(S/D/N) (S/G/R)(S/L/ G (M/I) T Y Y (A/P/S) T) (1888), (1886) D S V (K/Q) G with the (1887) proviso that if A at position 2, then not D or S in position 5 GYALDY (2038) GYIFSSY FPGSGS (1890) GYGNYLYFDV KASQSVSDDVA YAFKRYI QQNYNSPYT (1889) (1891) (1760) (1892) (1893) SYWIG KIFPGSGSADYNE DSEYSSGSGY SGDALTTQYAY KDTERPS QSADNSITYR (1894) NFKG (1895) (1896) (1897) (872) V (876) QRDSAWFAS RASESVDNSGISFMS ATSKLAS QQWISDPWT (2039) (1898) (1899) (1900) SGDALSEQYAY TASNQGS QQSKEVPWT (1901) (1902) (840) SGDALPKQYAY (869) SGDALPMQYGY (1903) RTSSSVNYMH (1904) GYRFTWY SAYNGN (1906) DVDYSSGSGY (1905) (1907) WYGIS WISAYNGNTNYAQ DAEYSLGSGY (1908) KLQG (861) (1909) GYRFSTF SAHNGN (1911) DAEYGSGSGY (1910) (1912) TFGIS WVSAHNGNTNYAQ DADYGSGSGY (1913) KLQG (1914) (1915) GYRFETY SYSGR (1917) DVDYGTGSGY (1916) (1918) GYRFRQY YISYSGRTSYNPS DVDYGSGSGY (1919) LTS (1920) (1921) QYGIS SGGGSD (1923) DAEYGSGSGY (1922) (1912) GYRFTRY TISGGGSDTYYPD GYALDY (1924) SVQG (1925) (2038) RYGIS QGYDVYSWFAY (1926) (1927) GYRFPHY (1928) HYGIS (1929) GYRFTRQ (1930) RQGIS (1931) GHSITSDY (1932) SDYAWN (1767) GFTFSTF (1933) TFGMS (1934) Group V GWSLTGPG IYGDGST AYEYAMDW WSVSTSGKSY LLS YHIRDLT (1935) (1936) (1937) (1938) (1939) (2040) Group W GYTFTSYW YPGSSL (1941) LSTGTFAY KSSQSLWDSTNQKNFL WTSTRES QNDYFYPLT IN (1942) T (1943) (1944) (1945) (1940) GYTFTSY NIYPGSSLTNYNE LTTGTFAY KSSQSLWDSGNQKNFL WTSYRES QNDYFYPHT (1946) KFKN (1947) (1948) T (1949) (1950) (1951) SYWIN YPGSSI (1953) LLTGTFAY KSSQSLLDSGNQKNFL WTS QNDYSYPLT (1952) (1954) T (1955) (T/Y) (1957) RES (1956) NIYPGSSITNYNE L(L/S)TGTFAY KSSQSL(W/L)DS(G/ QNDY(F/S)Y KFKN (1958) (1959) T)NQKNFLT (1960) P(L/H)T (1961) WPGSSL (1962) NIWPGSSLTNYNE KFKN (1963) NIYPGSSSTNYNE KFKN (1964) NI(Y/W)PGSS (L/I/S)TNYNEK FKN (1965) Group X GLTFSSSG IWYDGSKR ATNNDY RASQSVSSYLA TASNRAT QQYSNWPRT (1966) (1706) (1967) (704) (1968) (1969)

The ABs in the activatable antibodies of the disclosure specifically bind a PD-1 target, such as, for example, mammalian PD-1. In some embodiments, such Abs bind mammalian PD-1. In some embodiments, such Abs bind human PD-1. In some embodiments, such Abs bind non-human primate PD-1. Also included in the disclosure are ABs that bind to the same PD-1 epitope as an antibody of the disclosure and/or an activated activatable antibody described herein. Also included in the disclosure are ABs that compete with an anti-PD-1 antibody and/or an activated anti-PD-1 activatable antibody described herein for binding to a PD-1 target, e.g., human PD-1. Also included in the disclosure are ABs that cross-compete with an anti-PD-1 antibody and/or an activated anti-PD-1 activatable antibody described herein for binding to a PD-1 target, e.g., human PD-1.

The activatable anti-PD-1 antibodies provided herein include a masking moiety. In some embodiments, the masking moiety is an amino acid sequence that is coupled or otherwise attached to the anti-PD-1 antibody and is positioned within the activatable anti-PD-1 antibody construct such that the masking moiety reduces the ability of the anti-PD-1 antibody to specifically bind PD-1. Suitable masking moieties are identified using any of a variety of known techniques. For example, peptide masking moieties are identified using the methods described in PCT Publication No. WO 2009/025846 by Daugherty et al., the contents of which are hereby incorporated by reference in their entirety.

The activatable anti-PD-1 antibodies provided herein include a cleavable moiety. In some embodiments, the cleavable moiety includes an amino acid sequence that is a substrate for a protease, usually an extracellular protease. Suitable substrates are identified using any of a variety of known techniques. For example, peptide substrates are identified using the methods described in U.S. Pat. No. 7,666,817 by Daugherty et al.; in U.S. Pat. No. 8,563,269 by Stagliano et al.; and in PCT Publication No. WO 2014/026136 by La Porte et al., the contents of each of which are hereby incorporated by reference in their entirety. (See also Boulware et al. “Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics.” Biotechnol Bioeng. 106.3 (2010): 339-46).

Exemplary substrates include but are not limited to substrates cleavable by one or more of the following enzymes or proteases listed in Table 3.

TABLE 3 Exemplary Proteases and/or Enzymes ADAMS, ADAMTS, Cysteine proteinases, Serine proteases, e.g., e.g. ADAM8 e.g., Cruzipain activated protein C ADAM9 Legumain Cathepsin A ADAM 10 Otubain-2 Cathepsin G ADAM12 KLKs, e.g., Chymase ADAM15 KLK4  coagulation factor proteases ADAM17/TACE KLK5   (e.g., FVIIa, FIXa, FXa, ADAMDEC1 KLK6  FXIa, FXIIa) ADAMTS1 KLK7  Elastase ADAMTS4 KLK8  Granzyme B ADAMTS5 KLK10 Guanidinobenzoatase Aspartate proteases, KLK11 HtrA1 e.g., BACE KLK13 Human Neutrophil Elastase Renin KLK14 Lactoferrin Aspartic cathepsins, Metallo proteinases, Marapsin e.g., Cathepsin D e.g., Meprin NS3/4A Cathepsin E Neprilysin PACE4 Caspases, e.g., PSMA Plasmin Caspase 1 BMP-1 PSA Caspase 2 MMPs, e.g., tPA Caspase 3 MMP1  Thrombin Caspase 4 MMP2  Tryptase Caspase 5 MMP3  uPA Caspase 6 MMP7  Type II Transmembrane Caspase 7 MMP8  Serine Proteases (TTSPs), Caspase 8 MMP9  e.g., DESC1 Caspase 9 MMP10 DPP-4 Caspase 10 MMP11 FAP Caspase 14 MMP12 Hepsin Cysteine cathepsins, MMP13 Matriptase-2 e.g., Cathepsin B MMP14 MT-SP1/Matriptase Cathepsin C MMP15 TMPRSS2 Cathepsin K MMP16 TMPRSS3 Cathepsin L MMP17 TMPRSS4 Cathepsin S MMP19 Cathepsin V/L2 MMP20 Cathepsin X/Z/P MMP23 MMP24 MMP26 MMP27

The activatable anti-PD-1 antibodies described herein overcome a limitation of antibody therapeutics, particularly antibody therapeutics that are known to be toxic to at least some degree in vivo. Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. The activatable anti-PD-1 antibodies provided herein are designed to address the toxicity associated with the inhibition of the target in normal tissues by traditional therapeutic antibodies. These activatable anti-PD-1 antibodies remain masked until proteolytically activated at the site of disease. Starting with an anti-PD-1 antibody as a parental therapeutic antibody, the activatable anti-PD-1 antibodies of the invention were engineered by coupling the antibody to an inhibitory mask through a linker that incorporates a protease substrate.

When the AB is modified with a MM and is in the presence of the target, specific binding of the AB to its target is reduced or inhibited, as compared to the specific binding of the AB not modified with an MM or the specific binding of the parental AB to the target.

The K_(d) of the AB modified with a MM towards the target is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times greater than the K_(d) of the AB not modified with an MM or of the parental AB towards the target. Conversely, the binding affinity of the AB modified with a MM towards the target is at least 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times lower than the binding affinity of the AB not modified with an MM or of the parental AB towards the target.

In some embodiments, the dissociation constant (K_(d)) of the MM towards the AB is generally greater than the K_(d) of the AB towards the target. The K_(d) of the MM towards the AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times greater than the K_(d) of the AB towards the target. Conversely, the binding affinity of the MM towards the AB is generally lower than the binding affinity of the AB towards the target. The binding affinity of MM towards the AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times lower than the binding affinity of the AB towards the target.

In some embodiments, the dissociation constant (K_(d)) of the MM towards the AB is approximately equal to the K_(d) of the AB towards the target. In some embodiments, the dissociation constant (K_(d)) of the MM towards the AB is no more than the dissociation constant of the AB towards the target.

In some embodiments, the dissociation constant (K_(d)) of the MM towards the AB is less than the dissociation constant of the AB towards the target.

In some embodiments, the dissociation constant (K_(d)) of the MM towards the AB is greater than the dissociation constant of the AB towards the target.

In some embodiments, the MM has a K_(d) for binding to the AB that is no more than the K_(d) for binding of the AB to the target.

In some embodiments, the MM has a K_(d) for binding to the AB that is no less than the K_(d) for binding of the AB to the target.

In some embodiments, the MM has a K_(d) for binding to the AB that is approximately equal to the K_(d) for binding of the AB to the target.

In some embodiments, the MM has a K_(d) for binding to the AB that is less than the K_(d) for binding of the AB to the target.

In some embodiments, the MM has a K_(d) for binding to the AB that is greater than the K_(d) for binding of the AB to the target.

In some embodiments, the MM has a K_(d) for binding to the AB that is no more than 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 fold greater than the K_(d) for binding of the AB to the target. In some embodiments, the MM has a K_(d) for binding to the AB that is between 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1000, or 100-1,000 fold greater than the K_(d) for binding of the AB to the target.

In some embodiments, the MM has an affinity for binding to the AB that is less than the affinity of binding of the AB to the target.

In some embodiments, the MM has an affinity for binding to the AB that is no more than the affinity of binding of the AB to the target.

In some embodiments, the MM has an affinity for binding to the AB that is approximately equal of the affinity of binding of the AB to the target.

In some embodiments, the MM has an affinity for binding to the AB that is no less than the affinity of binding of the AB to the target.

In some embodiments, the MM has an affinity for binding to the AB that is greater than the affinity of binding of the AB to the target.

In some embodiments, the MM has an affinity for binding to the AB that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 less than the affinity of binding of the AB to the target. I In some embodiments, the MM has an affinity for binding to the AB that is between 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1000, or 100-1,000 fold less than the affinity of binding of the AB to the target. In some embodiments, the MM has an affinity for binding to the AB that is 2 to 20 fold less than the affinity of binding of the AB to the target. In some embodiments, a MM not covalently linked to the AB and at equimolar concentration to the AB does not inhibit the binding of the AB to the target.

When the AB is modified with a MM and is in the presence of the target specific binding of the AB to its target is reduced or inhibited, as compared to the specific binding of the AB not modified with an MM or the specific binding of the parental AB to the target. When compared to the binding of the AB not modified with an MM or the binding of the parental AB to the target the AB's ability to bind the target when modified with an MM can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more when measured in vivo or in an in vitro assay.

The MM inhibits the binding of the AB to the target. The MM binds the antigen binding domain of the AB and inhibits binding of the AB to the target. The MM can sterically inhibit the binding of the AB to the target. The MM can allosterically inhibit the binding of the AB to its target. In these embodiments when the AB is modified or coupled to a MM and in the presence of target there is no binding or substantially no binding of the AB to the target, or no more than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% binding of the AB to the target, as compared to the binding of the AB not modified with an MM, the parental AB, or the AB not coupled to an MM to the target, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer when measured in vivo or in an in vitro assay.

When an AB is coupled to or modified by a MM, the MM ‘masks’ or reduces or otherwise inhibits the specific binding of the AB to the target. When an AB is coupled to or modified by a MM, such coupling or modification can effect a structural change that reduces or inhibits the ability of the AB to specifically bind its target.

An AB coupled to or modified with an MM can be represented by the following formulae (in order from an amino (N) terminal region to carboxyl (C) terminal region:

(MM)-(AB)

(AB)-(MM)

(MM)-L-(AB)

(AB)-L-(MM)

where MM is a masking moiety, the AB is an antibody or antibody fragment thereof, and the L is a linker. In many embodiments, it can be desirable to insert one or more linkers, e.g., flexible linkers, into the composition so as to provide for flexibility.

In certain embodiments, the MM is not a natural binding partner of the AB. In some embodiments, the MM contains no or substantially no homology to any natural binding partner of the AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of the AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of the AB.

In some embodiments, the activatable antibodies include an AB that is modified by an MM and also includes one or more cleavable moieties (CM). Such activatable antibodies exhibit activatable/switchable binding, to the AB's target. Activatable antibodies generally include an antibody or antibody fragment (AB), modified by or coupled to a masking moiety (MM) and a modifiable or cleavable moiety (CM). In some embodiments, the CM contains an amino acid sequence that serves as a substrate for at least one protease.

The elements of the activatable antibodies are arranged so that the MM and CM are positioned such that in a cleaved (or relatively active) state and in the presence of a target, the AB binds a target while the activatable antibody is in an uncleaved (or relatively inactive) state in the presence of the target, specific binding of the AB to its target is reduced or inhibited. The specific binding of the AB to its target can be reduced due to the inhibition or masking of the AB's ability to specifically bind its target by the MM.

The K_(d) of the AB modified with a MM and a CM towards the target is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times greater than the K_(d) of the AB not modified with an MM and a CM or of the parental AB towards the target. Conversely, the binding affinity of the AB modified with a MM and a CM towards the target is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times lower than the binding affinity of the AB not modified with an MM and a CM or of the parental AB towards the target.

When the AB is modified with a MM and a CM and is in the presence of the target but not in the presence of a modifying agent (for example at least one protease), specific binding of the AB to its target is reduced or inhibited, as compared to the specific binding of the AB not modified with an MM and a CM or of the parental AB to the target. When compared to the binding of the parental AB or the binding of an AB not modified with an MM and a CM to its target, the AB's ability to bind the target when modified with an MM and a CM can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer when measured in vivo or in an in vitro assay.

As used herein, the term cleaved state refers to the condition of the activatable antibodies following modification of the CM by at least one protease. The term uncleaved state, as used herein, refers to the condition of the activatable antibodies in the absence of cleavage of the CM by a protease. As discussed above, the term “activatable antibodies” is used herein to refer to an activatable antibody in both its uncleaved (native) state, as well as in its cleaved state. It will be apparent to the ordinarily skilled artisan that in some embodiments a cleaved activatable antibody can lack an MM due to cleavage of the CM by protease, resulting in release of at least the MM (e.g., where the MM is not joined to the activatable antibodies by a covalent bond (e.g., a disulfide bond between cysteine residues).

By activatable or switchable is meant that the activatable antibody exhibits a first level of binding to a target when the activatable antibody is in a inhibited, masked or uncleaved state (i.e., a first conformation), and a second level of binding to the target in the uninhibited, unmasked and/or cleaved state (i.e., a second conformation), where the second level of target binding is greater than the first level of binding. In general, the access of target to the AB of the activatable antibody is greater in the presence of a cleaving agent capable of cleaving the CM, i.e., a protease, than in the absence of such a cleaving agent. Thus, when the activatable antibody is in the uncleaved state, the AB is inhibited from target binding and can be masked from target binding (i.e., the first conformation is such the AB cannot bind the target), and in the cleaved state the AB is not inhibited or is unmasked to target binding.

The CM and AB of the activatable antibodies are selected so that the AB represents a binding moiety for a given target, and the CM represents a substrate for a protease. In some embodiments, the protease is co-localized with the target at a treatment site or diagnostic site in a subject. As used herein, co-localized refers to being at the same site or relatively close nearby. In some embodiments, a protease cleaves a CM yielding an activated antibody that binds to a target located nearby the cleavage site. The activatable antibodies disclosed herein find particular use where, for example, a protease capable of cleaving a site in the CM, i.e., a protease, is present at relatively higher levels in or in close proximity to target-containing tissue of a treatment site or diagnostic site than in tissue of non-treatment sites (for example in healthy tissue). In some embodiments, a CM of the disclosure is also cleaved by one or more other proteases. In some embodiments, it is the one or more other proteases that is co-localized with the target and that is responsible for cleavage of the CM in vivo.

In some embodiments activatable antibodies provide for reduced toxicity and/or adverse side effects that could otherwise result from binding of the AB at non-treatment sites if the AB were not masked or otherwise inhibited from binding to the target.

In general, an activatable antibody can be designed by selecting an AB of interest and constructing the remainder of the activatable antibody so that, when conformationally constrained, the MM provides for masking of the AB or reduction of binding of the AB to its target. Structural design criteria can be to be taken into account to provide for this functional feature.

Activatable antibodies exhibiting a switchable phenotype of a desired dynamic range for target binding in an inhibited versus an uninhibited conformation are provided. Dynamic range generally refers to a ratio of (a) a maximum detected level of a parameter under a first set of conditions to (b) a minimum detected value of that parameter under a second set of conditions. For example, in the context of an activatable antibody, the dynamic range refers to the ratio of (a) a maximum detected level of target protein binding to an activatable antibody in the presence of at least one protease capable of cleaving the CM of the activatable antibodies to (b) a minimum detected level of target protein binding to an activatable antibody in the absence of the protease. The dynamic range of an activatable antibody can be calculated as the ratio of the dissociation constant of an activatable antibody cleaving agent (e.g., enzyme) treatment to the dissociation constant of the activatable antibodies cleaving agent treatment. The greater the dynamic range of an activatable antibody, the better the switchable phenotype of the activatable antibody. Activatable antibodies having relatively higher dynamic range values (e.g., greater than 1) exhibit more desirable switching phenotypes such that target protein binding by the activatable antibodies occurs to a greater extent (e.g., predominantly occurs) in the presence of a cleaving agent (e.g., enzyme) capable of cleaving the CM of the activatable antibodies than in the absence of a cleaving agent.

Activatable antibodies can be provided in a variety of structural configurations. Exemplary formulae for activatable antibodies are provided below. It is specifically contemplated that the N- to C-terminal order of the AB, MM and CM can be reversed within an activatable antibody. It is also specifically contemplated that the CM and MM can overlap in amino acid sequence, e.g., such that the CM is contained within the MM.

For example, activatable antibodies can be represented by the following formula (in order from an amino (N) terminal region to carboxyl (C) terminal region:

(MM)-(CM)-(AB)

(AB)-(CM)-(MM)

where MM is a masking moiety, CM is a cleavable moiety, and AB is an antibody or fragment thereof. It should be noted that although MM and CM are indicated as distinct components in the formulae above, in all exemplary embodiments (including formulae) disclosed herein it is contemplated that the amino acid sequences of the MM and the CM could overlap, e.g., such that the CM is completely or partially contained within the MM. In addition, the formulae above provide for additional amino acid sequences that can be positioned N-terminal or C-terminal to the activatable antibodies elements.

In certain embodiments, the MM is not a natural binding partner of the AB. In some embodiments, the MM contains no or substantially no homology to any natural binding partner of the AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of the AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of the AB.

In many embodiments it can be desirable to insert one or more linkers, e.g., flexible linkers, into the activatable antibody construct so as to provide for flexibility at one or more of the MM-CM junction, the CM-AB junction, or both. For example, the AB, MM, and/or CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, especially Gly and Ser, particularly Gly) to provide the desired flexibility. As such, the switchable phenotype of such activatable antibody constructs can benefit from introduction of one or more amino acids to provide for a flexible linker. In addition, as described below, where the activatable antibody is provided as a conformationally constrained construct, a flexible linker can be operably inserted to facilitate formation and maintenance of a cyclic structure in the uncleaved activatable antibody.

For example, in certain embodiments an activatable antibody comprises one of the following formulae (where the formula below represent an amino acid sequence in either N- to C-terminal direction or C- to N-terminal direction):

(MM)-L1-(CM)-(AB)

(MM)-(CM)-L2-(AB)

(MM)-L1-(CM)-L2-(AB)

wherein MM, CM, and AB are as defined above; wherein L1 and L2 are each independently and optionally present or absent, are the same or different flexible linkers that include at least 1 flexible amino acid (e.g., Gly). In addition, the formulae above provide for additional amino acid sequences that can be positioned N-terminal or C-terminal to the activatable antibodies elements. Examples include, but are not limited to, targeting moieties (e.g., a ligand for a receptor of a cell present in a target tissue) and serum half-life extending moieties (e.g., polypeptides that bind serum proteins, such as immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin (HAS)).

The CM is specifically cleaved by at least one protease at a rate of about 0.001-1500×10⁴M⁻¹S⁻¹ or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500×10⁴ M⁻¹S⁻¹. In some embodiments, the CM is specifically cleaved at a rate of about 100,000 M⁻¹S⁻¹. In some embodiments, the CM is specifically cleaved at a rate from about 1×10E2 to about 1×10E6 M⁻¹S⁻¹ (i.e., from about 1×10² to about 1×10⁶M⁻¹S⁻¹).

For specific cleavage by an enzyme, contact between the enzyme and CM is made. When the activatable antibody comprising an AB coupled to a MM and a CM is in the presence of target and sufficient enzyme activity, the CM can be cleaved. Sufficient enzyme activity can refer to the ability of the enzyme to make contact with the CM and effect cleavage. It can readily be envisioned that an enzyme can be in the vicinity of the CM but unable to cleave because of other cellular factors or protein modification of the enzyme.

Linkers suitable for use in compositions described herein are generally ones that provide flexibility of the modified AB or the activatable antibodies to facilitate the inhibition of the binding of the AB to the target. Such linkers are generally referred to as flexible linkers. Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS) n (SEQ ID NO: 363) and (GGGS)n (SEQ ID NO: 364), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore can be able to serve as a neutral tether between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to GGSG (SEQ ID NO: 365), GGSGG (SEQ ID NO: 366), GSGSG (SEQ ID NO: 367), GSGGG (SEQ ID NO: 368), GGGSG (SEQ ID NO: 369), GSSSG (SEQ ID NO: 370), and the like. The ordinarily skilled artisan will recognize that design of an activatable antibodies can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired activatable antibodies structure.

The disclosure also provides compositions and methods that include an activatable anti-PD-1 antibody that includes an antibody or antibody fragment (AB) that specifically binds PD-1, where the AB is coupled to a masking moiety (MM) that decreases the ability of the AB to bind its target. In some embodiments, the activatable anti-PD-1 antibody further includes a cleavable moiety (CM) that is a substrate for a protease. The compositions and methods provided herein enable the attachment of one or more agents to one or more cysteine residues in the AB without compromising the activity (e.g., the masking, activating or binding activity) of the activatable anti-PD-1 antibody. In some embodiments, the compositions and methods provided herein enable the attachment of one or more agents to one or more cysteine residues in the AB without reducing or otherwise disturbing one or more disulfide bonds within the MM. The compositions and methods provided herein produce an activatable anti-PD-1 antibody that is conjugated to one or more agents, e.g., any of a variety of therapeutic, diagnostic and/or prophylactic agents, for example, in some embodiments, without any of the agent(s) being conjugated to the MM of the activatable anti-PD-1 antibody. The compositions and methods provided herein produce conjugated activatable anti-PD-1 antibodies in which the MM retains the ability to effectively and efficiently mask the AB of the activatable antibody in an uncleaved state. The compositions and methods provided herein produce conjugated activatable anti-PD-1 antibodies in which the activatable antibody is still activated, i.e., cleaved, in the presence of a protease that can cleave the CM.

The activatable anti-PD-1 antibodies have at least one point of conjugation for an agent, but in the methods and compositions provided herein less than all possible points of conjugation are available for conjugation to an agent. In some embodiments, the one or more points of conjugation are sulfur atoms involved in disulfide bonds. In some embodiments, the one or more points of conjugation are sulfur atoms involved in interchain disulfide bonds. In some embodiments, the one or more points of conjugation are sulfur atoms involved in interchain sulfide bonds, but not sulfur atoms involved in intrachain disulfide bonds. In some embodiments, the one or more points of conjugation are sulfur atoms of cysteine or other amino acid residues containing a sulfur atom. Such residues can occur naturally in the antibody structure or can be incorporated into the antibody by site-directed mutagenesis, chemical conversion, or mis-incorporation of non-natural amino acids.

Also provided are methods of preparing a conjugate of an activatable anti-PD-1 antibody having one or more interchain disulfide bonds in the AB and one or more intrachain disulfide bonds in the MM, and a drug reactive with free thiols is provided. The method generally includes partially reducing interchain disulfide bonds in the activatable antibody with a reducing agent, such as, for example, TCEP; and conjugating the drug reactive with free thiols to the partially reduced activatable antibody. As used herein, the term partial reduction refers to situations where an activatable anti-PD-1 antibody is contacted with a reducing agent and less than all disulfide bonds, e.g., less than all possible sites of conjugation are reduced. In some embodiments, less than 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or less than 5% of all possible sites of conjugation are reduced.

In yet other embodiments, a method of reducing and conjugating an agent, e.g., a drug, to an activatable anti-PD-1 antibody resulting in selectivity in the placement of the agent is provided. The method generally includes partially reducing the activatable anti-PD-1 antibody with a reducing agent such that any conjugation sites in the masking moiety or other non-AB portion of the activatable antibody are not reduced, and conjugating the agent to interchain thiols in the AB. The conjugation site(s) are selected so as to allow desired placement of an agent to allow conjugation to occur at a desired site. The reducing agent is, for example, TCEP. The reduction reaction conditions such as, for example, the ratio of reducing agent to activatable antibody, the length of incubation, the temperature during the incubation, the pH of the reducing reaction solution, etc., are determined by identifying the conditions that produce a conjugated activatable antibody in which the MM retains the ability to effectively and efficiently mask the AB of the activatable antibody in an uncleaved state. The ratio of reduction agent to activatable anti-PD-1 antibody will vary depending on the activatable antibody. In some embodiments, the ratio of reducing agent to activatable anti-PD-1 antibody will be in a range from about 20:1 to 1:1, from about 10:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to 1:1, from about 7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to 1:1, from about 4:1 to 1:1, from about 3:1 to 1:1, from about 2:1 to 1:1, from about 20:1 to 1:1.5, from about 10:1 to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5, from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about 5:1 to 1:1.5, from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In some embodiments, the ratio is in a range of from about 5:1 to 1:1. In some embodiments, the ratio is in a range of from about 5:1 to 1.5:1. In some embodiments, the ratio is in a range of from about 4:1 to 1:1. In some embodiments, the ratio is in a range from about 4:1 to 1.5:1. In some embodiments, the ratio is in a range from about 8:1 to about 1:1. In some embodiments, the ratio is in a range of from about 2.5:1 to 1:1.

In some embodiments, a method of reducing interchain disulfide bonds in the AB of an activatable anti-PD-1 antibody and conjugating an agent, e.g., a thiol-containing agent such as a drug, to the resulting interchain thiols to selectively locate agent(s) on the AB is provided. The method generally includes partially reducing the AB with a reducing agent to form at least two interchain thiols without forming all possible interchain thiols in the activatable antibody; and conjugating the agent to the interchain thiols of the partially reduced AB. For example, the AB of the activatable antibody is partially reduced for about 1 hour at about 37° C. at a desired ratio of reducing agent:activatable antibody. In some embodiments, the ratio of reducing agent to activatable antibody will be in a range from about 20:1 to 1:1, from about 10:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to 1:1, from about 7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to 1:1, from about 4:1 to 1:1, from about 3:1 to 1:1, from about 2:1 to 1:1, from about 20:1 to 1:1.5, from about 10:1 to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5, from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about 5:1 to 1:1.5, from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In some embodiments, the ratio is in a range of from about 5:1 to 1:1. In some embodiments, the ratio is in a range of from about 5:1 to 1.5:1. In some embodiments, the ratio is in a range of from about 4:1 to 1:1. In some embodiments, the ratio is in a range from about 4:1 to 1.5:1. In some embodiments, the ratio is in a range from about 8:1 to about 1:1. In some embodiments, the ratio is in a range of from about 2.5:1 to 1:1.

The thiol-containing reagent can be, for example, cysteine or N-acetyl cysteine. The reducing agent can be, for example, TCEP. In some embodiments, the reduced activatable antibody can be purified prior to conjugation, using for example, column chromatography, dialysis, or diafiltration. Alternatively, the reduced antibody is not purified after partial reduction and prior to conjugation.

The invention also provides partially reduced activatable anti-PD-1 antibodies in which at least one interchain disulfide bond in the activatable antibody has been reduced with a reducing agent without disturbing any intrachain disulfide bonds in the activatable antibody, wherein the activatable antibody includes an antibody or an antigen binding fragment thereof (AB) that specifically binds to PD-1, a masking moiety (MM) that inhibits the binding of the AB of the activatable antibody in an uncleaved state to the PD-1 target, and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. In some embodiments the MM is coupled to the AB via the CM. In some embodiments, one or more intrachain disulfide bond(s) of the activatable antibody is not disturbed by the reducing agent. In some embodiments, one or more intrachain disulfide bond(s) of the MM within the activatable antibody is not disturbed by the reducing agent. In some embodiments, the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM. In some embodiments, reducing agent is TCEP.

The disclosure also provides partially reduced activatable antibodies in which at least one interchain disulfide bond in the activatable antibody has been reduced with a reducing agent without disturbing any intrachain disulfide bonds in the activatable antibody, wherein the activatable antibody includes an antibody or an antigen binding fragment thereof (AB) that specifically binds to the target, e.g., PD-1, a masking moiety (MM) that inhibits the binding of the AB of the activatable antibody in an uncleaved state to the target, and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for at least one protease. In some embodiments, the MM is coupled to the AB via the CM. In some embodiments, one or more intrachain disulfide bond(s) of the activatable antibody is not disturbed by the reducing agent. In some embodiments, one or more intrachain disulfide bond(s) of the MM within the activatable antibody is not disturbed by the reducing agent. In some embodiments, the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM. In some embodiments, reducing agent is TCEP.

In some embodiments, the activatable antibodies described herein also include an agent conjugated to the activatable antibody. In some embodiments, the conjugated agent is a therapeutic agent, such as an anti-inflammatory. In such embodiments, the agent is conjugated to a carbohydrate moiety of the activatable antibody, for example, in some embodiments, where the carbohydrate moiety is located outside the antigen-binding region of the antibody or antigen-binding fragment in the activatable antibody. In some embodiments, the agent is conjugated to a sulfhydryl group of the antibody or antigen-binding fragment in the activatable antibody.

In some embodiments, the agent is a detectable moiety such as, for example, a label or other marker. For example, the agent is or includes a radiolabeled amino acid, one or more biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzymatic labels, and/or one or more chemiluminescent agents. In some embodiments, detectable moieties are attached by spacer molecules.

Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies of the disclosure. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

Coupling can be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. In some embodiments, the binding is, however, covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present disclosure, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987).

In some embodiments, in addition to the compositions and methods provided herein, the conjugated activatable antibody can also be modified for site-specific conjugation through modified amino acid sequences inserted or otherwise included in the activatable antibody sequence. These modified amino acid sequences are designed to allow for controlled placement and/or dosage of the conjugated agent within a conjugated activatable antibody. For example, the activatable antibody can be engineered to include cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not negatively impact protein folding and assembly, nor alter antigen binding. In some embodiments, the activatable antibody can be engineered to include or otherwise introduce one or more non-natural amino acid residues within the activatable antibody to provide suitable sites for conjugation. In some embodiments, the activatable antibody can be engineered to include or otherwise introduce enzymatically activatable peptide sequences within the activatable antibody sequence.

Suitable linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat. No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. In some embodiments, suitable linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC. Additional linkers include, but are not limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB.

The linkers described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NETS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.

In some embodiments, the linkers are cleavable. In some embodiments, the linkers are non-cleavable. In some embodiments, two or more linkers are present. The two or more linkers are all the same, i.e., cleavable or non-cleavable, or the two or more linkers are different, i.e., at least one cleavable and at least one non-cleavable.

The present disclosure utilizes several methods for attaching agents to ABs: (a) attachment to the carbohydrate moieties of the AB, or (b) attachment to sulfhydryl groups of the AB, or (c) attachment to amino groups of the AB, or (d) attachment to carboxylate groups of the AB. According to the disclosure, ABs can be covalently attached to an agent through an intermediate linker having at least two reactive groups, one to react with AB and one to react with the agent. The linker, which can include any compatible organic compound, can be chosen such that the reaction with AB (or agent) does not adversely affect AB reactivity and selectivity. Furthermore, the attachment of linker to agent might not destroy the activity of the agent. Suitable linkers for reaction with oxidized antibodies or oxidized antibody fragments include those containing an amine selected from the group consisting of primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide groups. Such reactive functional groups can exist as part of the structure of the linker, or can be introduced by suitable chemical modification of linkers not containing such groups.

According to the present disclosure, suitable linkers for attachment to reduced ABs include those having certain reactive groups capable of reaction with a sulfhydryl group of a reduced antibody or fragment. Such reactive groups include, but are not limited to: reactive haloalkyl groups (including, for example, haloacetyl groups), p-mercuribenzoate groups and groups capable of Michael-type addition reactions (including, for example, maleimides and groups of the type described by Mitra and Lawton, 1979, J. Amer. Chem. Soc. 101: 3097-3110).

According to the present disclosure, suitable linkers for attachment to neither oxidized nor reduced Abs include those having certain functional groups capable of reaction with the primary amino groups present in unmodified lysine residues in the Ab. Such reactive groups include, but are not limited to, NHS carboxylic or carbonic esters, sulfo-NHS carboxylic or carbonic esters, 4-nitrophenyl carboxylic or carbonic esters, pentafluorophenyl carboxylic or carbonic esters, acyl imidazoles, isocyanates, and isothiocyanates.

According to the present disclosure, suitable linkers for attachment to neither oxidized nor reduced Abs include those having certain functional groups capable of reaction with the carboxylic acid groups present in aspartate or glutamate residues in the Ab, which have been activated with suitable reagents. Suitable activating reagents include EDC, with or without added NHS or sulfo-NHS, and other dehydrating agents utilized for carboxamide formation. In these instances, the functional groups present in the suitable linkers would include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides.

The agent can be attached to the linker before or after the linker is attached to the AB. In certain applications it can be desirable to first produce an AB-linker intermediate in which the linker is free of an associated agent. Depending upon the particular application, a specific agent can then be covalently attached to the linker. In some embodiments, the AB is first attached to the MM, CM and associated linkers and then attached to the linker for conjugation purposes.

Branched Linkers:

In specific embodiments, branched linkers that have multiple sites for attachment of agents are utilized. For multiple site linkers, a single covalent attachment to an AB would result in an AB-linker intermediate capable of binding an agent at a number of sites. The sites can be aldehyde or sulfhydryl groups or any chemical site to which agents can be attached.

In some embodiments, higher specific activity (or higher ratio of agents to AB) can be achieved by attachment of a single site linker at a plurality of sites on the AB. This plurality of sites can be introduced into the AB by either of two methods. First, one can generate multiple aldehyde groups and/or sulfhydryl groups in the same AB. Second, one can attach to an aldehyde or sulfhydryl of the AB a “branched linker” having multiple functional sites for subsequent attachment to linkers. The functional sites of the branched linker or multiple site linker can be aldehyde or sulfhydryl groups, or can be any chemical site to which linkers can be attached. Still higher specific activities can be obtained by combining these two approaches, that is, attaching multiple site linkers at several sites on the AB.

Cleavable Linkers:

Peptide linkers that are susceptible to cleavage by enzymes of the complement system, such as but not limited to u-plasminogen activator, tissue plasminogen activator, trypsin, plasmin, or another enzyme having proteolytic activity can be used in one embodiment of the present disclosure. According to one method of the present disclosure, an agent is attached via a linker susceptible to cleavage by complement. The antibody is selected from a class that can activate complement. The antibody-agent conjugate, thus, activates the complement cascade and releases the agent at the target site. According to another method of the present disclosure, an agent is attached via a linker susceptible to cleavage by enzymes having a proteolytic activity such as a u-plasminogen activator, a tissue plasminogen activator, plasmin, or trypsin.

Non-limiting examples of cleavable linker sequences are provided in Table 5.

TABLE 5 Exemplary Linker Sequences for Conjugation Types of Cleavable Sequences Amino Acid Sequence Plasmin cleavable sequences Pro-urokinase PRFKIIGG (SEQ ID NO: 587) PRFRIIGG (SEQ ID NO: 588) TGFβ SSRHRRALD (SEQ ID NO: 589) Plasminogen RKSSIIIRMRDVVL (SEQ ID NO: 590) Staphylokinase SSSFDKGKYKKGDDA (SEQ ID NO: 591) SSSFDKGKYKRGDDA (SEQ ID NO: 592) Factor Xa cleavable sequences IEGR (SEQ ID NO: 593) IDGR (SEQ ID NO: 594) GGSIDGR (SEQ ID NO: 595) MMP cleavable sequences Gelatinase A PLGLWA (SEQ ID NO: 596) Collagenase cleavable sequences Calf skin collagen (α1(I) chain) GPQGIAGQ (SEQ ID NO: 597) Calf skin collagen (α2(I) chain) GPQGLLGA (SEQ ID NO: 598) Bovine cartilage collagen (α1(II) chain) GIAGQ (SEQ ID NO: 599) Human liver collagen (α1(III) chain) GPLGIAGI (SEQ ID NO: 600) Human α₂M GPEGLRVG (SEQ ID NO: 601) Human PZP YGAGLGVV (SEQ ID NO: 602) AGLGVVER (SEQ ID NO: 603) AGLGISST (SEQ ID NO: 604) Rat α₁M EPQALAMS (SEQ ID NO: 605) QALAMSAI (SEQ ID NO: 606) Rat α₂M AAYHLVSQ (SEQ ID NO: 607) MDAFLESS (SEQ ID NO: 608) Rat α₁I₃(2J) ESLPVVAV (SEQ ID NO: 609) Rat α₁I₃(27J) SAPAVESE (SEQ ID NO: 610) Human fibroblast collagenase DVAQFVLT (SEQ ID NO: 611) (autolytic cleavages) VAQFVLTE (SEQ ID NO: 612) AQFVLTEG (SEQ ID NO: 613) PVQPIGPQ (SEQ ID NO: 614)

In addition, agents can be attached via disulfide bonds (for example, the disulfide bonds on a cysteine molecule) to the AB. Since many tumors naturally release high levels of glutathione (a reducing agent) this can reduce the disulfide bonds with subsequent release of the agent at the site of delivery. In some embodiments, the reducing agent that would modify a CM would also modify the linker of the conjugated activatable antibody.

Spacers and Cleavable Elements: In some embodiments, it can be necessary to construct the linker in such a way as to optimize the spacing between the agent and the AB of the activatable antibody. This can be accomplished by use of a linker of the general structure:

W—(CH₂)_(n)-Q

wherein W is either —NH—CH₂— or —CH₂—; Q is an amino acid, peptide; and n is an integer from 0 to 20.

In some embodiments, the linker can comprise a spacer element and a cleavable element. The spacer element serves to position the cleavable element away from the core of the AB such that the cleavable element is more accessible to the enzyme responsible for cleavage. Certain of the branched linkers described above can serve as spacer elements.

Throughout this discussion, it should be understood that the attachment of linker to agent (or of spacer element to cleavable element, or cleavable element to agent) need not be particular mode of attachment or reaction. Any reaction providing a product of suitable stability and biological compatibility is acceptable.

Serum Complement and Selection of Linkers:

According to one method of the present disclosure, when release of an agent is desired, an AB that is an antibody of a class that can activate complement is used. The resulting conjugate retains both the ability to bind antigen and activate the complement cascade. Thus, according to this embodiment of the present disclosure, an agent is joined to one end of the cleavable linker or cleavable element and the other end of the linker group is attached to a specific site on the AB. For example, if the agent has an hydroxy group or an amino group, it can be attached to the carboxy terminus of a peptide, amino acid or other suitably chosen linker via an ester or amide bond, respectively. For example, such agents can be attached to the linker peptide via a carbodimide reaction. If the agent contains functional groups that would interfere with attachment to the linker, these interfering functional groups can be blocked before attachment and deblocked once the product conjugate or intermediate is made. The opposite or amino terminus of the linker is then used either directly or after further modification for binding to an AB that is capable of activating complement.

Linkers (or spacer elements of linkers) can be of any desired length, one end of which can be covalently attached to specific sites on the AB of the activatable antibody. The other end of the linker or spacer element can be attached to an amino acid or peptide linker.

Thus when these conjugates bind to antigen in the presence of complement the amide or ester bond that attaches the agent to the linker will be cleaved, resulting in release of the agent in its active form. These conjugates, when administered to a subject, will accomplish delivery and release of the agent at the target site, and are particularly effective for the in vivo delivery of pharmaceutical agents, antibiotics, antimetabolites, antiproliferative agents and the like as presented in but not limited to those in Table 5.

Linkers for Release without Complement Activation:

In yet another application of targeted delivery, release of the agent without complement activation is desired since activation of the complement cascade will ultimately lyse the target cell. Hence, this approach is useful when delivery and release of the agent should be accomplished without killing the target cell. Such is the goal when delivery of cell mediators such as hormones, enzymes, corticosteroids, neurotransmitters, genes or enzymes to target cells is desired. These conjugates can be prepared by attaching the agent to an AB that is not capable of activating complement via a linker that is mildly susceptible to cleavage by serum proteases. When this conjugate is administered to an individual, antigen-antibody complexes will form quickly whereas cleavage of the agent will occur slowly, thus resulting in release of the compound at the target site.

Biochemical Cross Linkers:

In some embodiments, the activatable antibody can be conjugated to one or more therapeutic agents using certain biochemical cross-linkers. Cross-linking reagents form molecular bridges that tie together functional groups of two different molecules. To link two different proteins in a step-wise manner, hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.

Peptidyl linkers cleavable by lysosomal proteases are also useful, for example, Val-Cit, Val-Ala or other dipeptides. In addition, acid-labile linkers cleavable in the low-pH environment of the lysosome can be used, for example: bis-sialyl ether. Other suitable linkers include cathepsin-labile substrates, particularly those that show optimal function at an acidic pH.

Exemplary hetero-bifunctional cross-linkers are referenced in Table 6.

TABLE 6 Exemplary Hetero-Bifunctional Cross Linkers HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm Length after Reactive Advantages and cross-linking Linker Toward Applications (Angstroms) SMPT Primary amines Greater stability 11.2 Å Sulfhydryls SPDP Primary amines Thiolation  6.8 Å Sulfhydryls Cleavable cross-linking LC- Primary amines Extended spacer arm 15.6 Å SPDP Sulfhydryls Sulfo- Primary amines Extender spacer arm 15.6 Å LC- Sulfhydryls Water-soluble SPDP SMCC Primary amines Stable maleimide reactive 11.6 Å group Sulfhydryls Enzyme-antibody conjugation Hapten-carrier protein conjugation Sulfo- Primary amines Stable maleimide reactive 11.6 Å SMCC group Sulfhydryls Water-soluble Enzyme-antibody conjugation MBS Primary amines Enzyme-antibody conjugation  9.9 Å Sulfhydryls Hapten-carrier protein conjugation Sulfo- Primary amines Water-soluble  9.9 Å MBS Sulfhydryls SIAB Primary amines Enzyme-antibody conjugation 10.6 Å Sulfhydryls Sulfo- Primary amines Water-soluble 10.6 Å SIAB Sulfhydryls SMPB Primary amines Extended spacer arm 14.5 Å Sulfhydryls Enzyme-antibody conjugation Sulfo- Primary amines Extended spacer arm 14.5 Å SMPB Sulfhydryls Water-soluble EDE/ Primary amines Hapten-Carrier conjugation 0 Sulfo- Carboxyl groups NHS ABH Carbohydrates Reacts with sugar groups 11.9 Å Nonselective

Non-Cleavable Linkers or Direct Attachment:

In some embodiments of the disclosure, the conjugate can be designed so that the agent is delivered to the target but not released. This can be accomplished by attaching an agent to an AB either directly or via a non-cleavable linker.

These non-cleavable linkers can include amino acids, peptides, D-amino acids or other organic compounds that can be modified to include functional groups that can subsequently be utilized in attachment to ABs by the methods described herein. A-general formula for such an organic linker could be

W—(CH₂)_(n)-Q

wherein W is either —NH—CH₂— or —CH₂—; Q is an amino acid, peptide; and n is an integer from 0 to 20.

Non-Cleavable Conjugates:

In some embodiments, a compound can be attached to ABs that do not activate complement. When using ABs that are incapable of complement activation, this attachment can be accomplished using linkers that are susceptible to cleavage by activated complement or using linkers that are not susceptible to cleavage by activated complement.

The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab′ fragments of the antibody of the present disclosure can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Definitions

Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The term “a” entity or “an” entity refers to one or more of that entity. For example, a compound refers to one or more compounds. As such, the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

As used herein, the term “antibody” refers to immunoglobulin molecules and antigen-binding portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. By “specifically bind” or “immunoreacts with” or “immunospecifically bind” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, domain antibody, single chain, Fab, and F(ab′)₂ fragments, scFvs, and an Fab expression library.

The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG₁, IgG₂, and others. Furthermore, in humans, the light chain can be a kappa chain or a lambda chain.

The term “monoclonal antibody” (mAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

The term “antigen-binding site” or “binding portion” refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as “hypervariable regions,” are interposed between more conserved flanking stretches known as “framework regions,” or “FRs”. Thus, the term “FR” refers to amino acid sequences that are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin, an scFv, or a T-cell receptor. The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N-terminal or C-terminal peptides of a polypeptide. An antibody is said to specifically bind an antigen when the dissociation constant is ≤1 μM; in some embodiments, ≤100 nM and in some embodiments, ≤10 nM.

As used herein, the terms “specific binding,” “immunological binding,” and “immunological binding properties” refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K_(d)) of the interaction, wherein a smaller K_(d) represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (K_(on)) and the “off rate constant” (K_(off)) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) is equal to the dissociation constant K_(d). (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the present disclosure is said to specifically bind to the target, when the binding constant (K_(d)) is ≤1 μM, in some embodiments ≤100 nM, in some embodiments 10 nM, and in some embodiments ≤100 μM to about 1 μM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

The term “isolated polynucleotide” as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence. Polynucleotides in accordance with the disclosure include the nucleic acid molecules encoding the heavy chain immunoglobulin molecules shown herein, and nucleic acid molecules encoding the light chain immunoglobulin molecules shown herein.

The term “isolated protein” referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the “isolated protein” (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g., free of murine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature.

The term “polypeptide” is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein fragments, and analogs are species of the polypeptide genus. Polypeptides in accordance with the disclosure comprise the heavy chain immunoglobulin molecules shown herein, and the light chain immunoglobulin molecules shown herein, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof.

The term “naturally-occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions of components so described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

The term “control sequence” as used herein refers to polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. The term “polynucleotide” as referred to herein means nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

The term oligonucleotide referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset generally comprising a length of 200 bases or fewer. In some embodiments, oligonucleotides are 10 to 60 bases in length and in some embodiments, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes, although oligonucleotides can be double stranded, e.g., for use in the construction of a gene mutant. Oligonucleotides of the disclosure are either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” referred to herein includes nucleotides with modified or substituted sugar groups and the like. The term “oligonucleotide linkages” referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselerloate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti Cancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotide can include a label for detection, if desired.

As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd Edition, E. S. Golub and D. R. Green, Eds., Sinauer Associates, Sunderland, Mass. (1991)). Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids can also be suitable components for polypeptides of the present disclosure. Examples of unconventional amino acids include: 4 hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5′ end the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5′ direction. The direction of 5′ to 3′ addition of nascent RNA transcripts is referred to as the transcription direction sequence regions on the DNA strand having the same sequence as the RNA and that are 5′ to the 5′ end of the RNA transcript are referred to as “upstream sequences”, sequence regions on the DNA strand having the same sequence as the RNA and that are 3′ to the 3′ end of the RNA transcript are referred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, in some embodiments, at least 90 percent sequence identity, in some embodiments, at least 95 percent sequence identity, and in some embodiments, at least 99 percent sequence identity.

In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, in some embodiments, at least 80%, 90%, 95%, and in some embodiments, 99%. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Suitable amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. In some embodiments, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the disclosure.

Suitable amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (for example, conservative amino acid substitutions) can be made in the naturally-occurring sequence (for example, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptide that has an amino terminal and/or carboxy-terminal deletion and/or one or more internal deletion(s), but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full length cDNA sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, in some embodiments, at least 14 amino acids long, in some embodiments, at least 20 amino acids long, usually at least 50 amino acids long, and in some embodiments, at least 70 amino acids long. The term “analog” as used herein refers to polypeptides that are comprised of a segment of at least 25 amino acids that has substantial identity to a portion of a deduced amino acid sequence and that has specific binding to the target, under suitable binding conditions. Typically, polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally-occurring sequence. Analogs typically are at least 20 amino acids long, in some embodiments, at least 50 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide.

The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

As used herein, the terms “label” or “labeled” refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), or radionuclides (e.g., fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term “pharmaceutical agent or drug” as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and in some embodiments, a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.

Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, in some embodiments, more than about 85%, 90%, 95%, and 99%. In some embodiments, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

The term patient includes human and veterinary subjects.

Antibodies and/or activatable antibodies of the disclosure specifically bind a given target, e.g., a human target protein such as human PD-1. Also included in the disclosure are antibodies and/or activatable antibodies that bind to the same epitope as the antibodies and/or activatable antibodies described herein. Also included in the disclosure are antibodies and/or antibodies activatable antibodies that compete with an anti-PD-1 antibody and/or an anti-PD-1 activatable antibody described herein for binding to PD-1, e.g., human PD-1. Also included in the disclosure are antibodies and/or antibodies activatable antibodies that cross-compete with an anti-PD-1 antibody and/or an anti-PD-1 activatable antibody described herein for binding to PD-1, e.g., human PD-1.

Those skilled in the art will recognize that it is possible to determine, without undue experimentation, if a monoclonal antibody (e.g., a murine monoclonal or humanized antibody) has the same specificity as a monoclonal antibody used in the methods described herein by ascertaining whether the former prevents the latter from binding to the target. If the monoclonal antibody being tested competes with the monoclonal antibody of the disclosure, as shown by a decrease in binding by the monoclonal antibody of the disclosure, then the two monoclonal antibodies bind to the same, or a closely related, epitope. An alternative method for determining whether a monoclonal antibody has the specificity of a monoclonal antibody of the disclosure is to pre-incubate the monoclonal antibody of the disclosure with the target and then add the monoclonal antibody being tested to determine if the monoclonal antibody being tested is inhibited in its ability to bind the target. If the monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody of the disclosure.

Multispecific Activatable Antibodies

The disclosure also provides multispecific anti-PD-1 activatable antibodies. The multispecific activatable antibodies provided herein are multispecific antibodies that recognize PD-1 and at least one or more different antigens or epitopes and that include at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the multispecific antibody such that coupling of the MM reduces the ability of the antigen- or epitope-binding domain to bind its target. In some embodiments, the MM is coupled to the antigen- or epitope-binding domain of the multispecific antibody via a cleavable moiety (CM) that functions as a substrate for at least one protease. The activatable multispecific antibodies provided herein are stable in circulation, activated at intended sites of therapy and/or diagnosis but not in normal, i.e., healthy tissue, and, when activated, exhibit binding to a target that is at least comparable to the corresponding, unmodified multispecific antibody.

In some embodiments, the multispecific activatable antibodies are designed to engage immune effector cells, also referred to herein as immune-effector cell engaging multispecific activatable antibodies. In some embodiments, the multispecific activatable antibodies are designed to engage leukocytes, also referred to herein as leukocyte engaging multispecific activatable antibodies. In some embodiments, the multispecific activatable antibodies are designed to engage T cells, also referred to herein as T-cell engaging multispecific activatable antibodies. In some embodiments, the multispecific activatable antibodies engage a surface antigen on a leukocyte, such as on a T cell, on a natural killer (NK) cell, on a myeloid mononuclear cell, on a macrophage, and/or on another immune effector cell. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is a NK cell. In some embodiments, the immune effector cell is a mononuclear cell, such as a myeloid mononuclear cell. In some embodiments, the multispecific activatable antibodies are designed to bind or otherwise interact with more than one target and/or more than one epitope, also referred to herein as multi-antigen targeting activatable antibodies. As used herein, the terms “target” and “antigen” are used interchangeably.

In some embodiments, immune effector cell engaging multispecific activatable antibodies of the disclosure include a targeting antibody or antigen-binding fragment thereof that binds PD-1 and an immune effector cell engaging antibody or antigen-binding portion thereof, where at least one of the targeting antibody or antigen-binding fragment thereof and/or the immune effector cell engaging antibody or antigen-binding portion thereof is masked. In some embodiments, the immune effector cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen-binding fragment thereof (AB1) that binds a first, immune effector cell engaging target, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind the first target. In some embodiments, the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds PD-1, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind PD-1. In some embodiments, the immune effector cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen-binding fragment thereof (AB1) that binds a first, immune effector cell engaging target, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MIMI reduces the ability of the AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds PD-1, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind PD-1. In some embodiments, the non-immune effector cell engaging antibody is a cancer targeting antibody. In some embodiments the non-immune cell effector antibody is an IgG. In some embodiments the immune effector cell engaging antibody is a scFv. In some embodiments the PD-1-targeting antibody (e.g., non-immune cell effector antibody) is an IgG and the immune effector cell engaging antibody is a scFv. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is a NK cell. In some embodiments, the immune effector cell is a myeloid mononuclear cell.

In some embodiments, T-cell engaging multispecific activatable antibodies of the disclosure include a PD-1-targeting antibody or antigen-binding fragment thereof and a T-cell engaging antibody or antigen-binding portion thereof, where at least one of the PD-1-targeting antibody or antigen-binding fragment thereof and/or the T-cell engaging antibody or antigen-binding portion thereof is masked. In some embodiments, the T-cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen-binding fragment thereof (AB1) that binds a first, T-cell engaging target, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind the first target. In some embodiments, the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds PD-1, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind PD-1. In some embodiments, the T-cell engaging antibody or antigen binding fragment thereof includes a first antibody or antigen-binding fragment thereof (AB1) that binds a first, T-cell engaging target, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind the first target, and the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds PD-1, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind PD-1.

In some embodiments of an immune effector cell engaging multispecific activatable antibody, one antigen is PD-1, and another antigen is typically a stimulatory or inhibitory receptor present on the surface of a T-cell, natural killer (NK) cell, myeloid mononuclear cell, macrophage, and/or other immune effector cell, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, TIGIT, TIM3, or VISTA. In some embodiments, the antigen is a stimulatory receptor present on the surface of a T cell or NK cell; examples of such stimulatory receptors include, but are not limited to, CD3, CD27, CD28, CD137 (also referred to as 4-1BB), GITR, HVEM, ICOS, NKG2D, and OX40. In some embodiments, the antigen is an inhibitory receptor present on the surface of a T-cell; examples of such inhibitory receptors include, but are not limited to, BTLA, CTLA-4, LAG3, TIGIT, TIM3, and NK-expressed KIRs. The antibody domain conferring specificity to the T-cell surface antigen can also be substituted by a ligand or ligand domain that binds to a T-cell receptor, a NK-cell receptor, a macrophage receptor, and/or other immune effector cell receptor, such as, but not limited to, B7-1, B7-2, B7H3, PDL1, PDL2, or TNFSF9.

In some embodiments, the T-cell engaging multispecific activatable antibody includes an anti-CD3 epsilon (CD3ε, also referred to herein as CD3e and CD3) scFv and a targeting antibody or antigen-binding fragment thereof, where at least one of the anti-CD3ε scFv and/or the targeting antibody or antigen-binding portion thereof is masked. In some embodiments, the CD3ε scFv includes a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind CD3ε. In some embodiments, the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds PD-1, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind PD-1. In some embodiments, the CD3ε scFv includes a first antibody or antigen-binding fragment thereof (AB1) that binds CD3ε, where the AB1 is attached to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind CD3ε, and the targeting antibody or antigen-binding fragment thereof includes a second antibody or fragment thereof that includes a second antibody or antigen-binding fragment thereof (AB2) that binds PD-1, where the AB2 is attached to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind PD-1.

In some embodiments, the multi-antigen targeting antibodies and/or multi-antigen targeting activatable antibodies include at least a first antibody or antigen-binding fragment thereof that binds a first target and/or first epitope and a second antibody or antigen-binding fragment thereof that binds a second target and/or a second epitope. In some embodiments, the multi-antigen targeting antibodies and/or multi-antigen targeting activatable antibodies bind two or more different targets. In some embodiments, the multi-antigen targeting antibodies and/or multi-antigen targeting activatable antibodies bind two or more different epitopes on the same target. In some embodiments, the multi-antigen targeting antibodies and/or multi-antigen targeting activatable antibodies bind a combination of two or more different targets and two or more different epitopes on the same target.

In some embodiments, a multispecific activatable antibody comprising an IgG has the IgG variable domains masked. In some embodiments, a multispecific activatable antibody comprising a scFv has the scFv domains masked. In some embodiments, a multispecific activatable antibody has both IgG variable domains and scFv domains, where at least one of the IgG variable domains is coupled to a masking moiety. In some embodiments, a multispecific activatable antibody has both IgG variable domains and scFv domains, where at least one of the scFv domains is coupled to a masking moiety. In some embodiments, a multispecific activatable antibody has both IgG variable domains and scFv domains, where at least one of the IgG variable domains is coupled to a masking moiety and at least one of the scFv domains is coupled to a masking moiety. In some embodiments, a multispecific activatable antibody has both IgG variable domains and scFv domains, where each of the IgG variable domains and the scFv domains is coupled to its own masking moiety. In some embodiments, one antibody domain of a multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for a T-cell surface antigen. In some embodiments, one antibody domain of a multispecific activatable antibody has specificity for a target antigen and another antibody domain has specificity for another target antigen. In some embodiments, one antibody domain of a multispecific activatable antibody has specificity for an epitope of a target antigen and another antibody domain has specificity for another epitope of the target antigen.

In a multispecific activatable antibody, a scFv can be fused to the carboxyl terminus of the heavy chain of an IgG activatable antibody, to the carboxyl terminus of the light chain of an IgG activatable antibody, or to the carboxyl termini of both the heavy and light chains of an IgG activatable antibody. In a multispecific activatable antibody, a scFv can be fused to the amino terminus of the heavy chain of an IgG activatable antibody, to the amino terminus of the light chain of an IgG activatable antibody, or to the amino termini of both the heavy and light chains of an IgG activatable antibody. In a multispecific activatable antibody, a scFv can be fused to any combination of one or more carboxyl termini and one or more amino termini of an IgG activatable antibody. In some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is attached to and masks an antigen binding domain of the IgG. In some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is attached to and masks an antigen binding domain of at least one scFv. In some embodiments, a masking moiety (MM) linked to a cleavable moiety (CM) is attached to and masks an antigen binding domain of an IgG and a masking moiety (MM) linked to a cleavable moiety (CM) is attached to and masks an antigen binding domain of at least one scFv.

The disclosure provides examples of multispecific activatable antibody structures which include, but are not limited to, the following: (VL-CL)₂:(VH-CH1-CH2-CH3-L4-VH*-L3-VL*-L2-CM-L1-MM)₂; (VL-CL)₂:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*-L2-CM-L1-MM)₂; (MM-L1-CM-L2-VL-CL)₂:(VH-CH1-CH2-CH3-L4-VH*-L3-VL*)₂; (MM-L1-CM-L2-VL-CL)₂:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*)₂; (VL-CL)₂:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; (VL-CL)₂:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂; (MM-L1-CM-L2-VL-CL)₂:(VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; (MM-L1-CM-L2-VL-CL)₂:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂: (VH-CH1-CH2-CH3)₂; (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂: (VH-CH1-CH2-CH3)₂; (MM-L1-CM-L2-VL*-L3-VH*-L4-VL-CL)₂:(VH-CH1-CH2-CH3)₂; (MM-L1-CM-L2-VH*-L3-VL*-L4-VL-CL)₂:(VH-CH1-CH2-CH3)₂; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂: (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂: (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂: (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂: (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VH*-L3-VL*)₂: (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VH*-L3-VL*)₂: (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VL*-L3-VH*)₂: (MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VL*-L3-VH*)₂: (MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂: (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM)₂: (VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂; (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂: (VL*-L3-VH*-L4-VH-CH1-CH2-CH3)₂; or (VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM)₂: (VH*-L3-VL*-L4-VH-CH1-CH2-CH3)₂, wherein: VL and VH represent the light and heavy variable domains of the first specificity, contained in the IgG; VL* and VH* represent the variable domains of the second specificity, contained in the scFv; L1 is a linker peptide connecting the masking moiety (MM) and the cleavable moiety (CM); L2 is a linker peptide connecting the cleavable moiety (CM), and the antibody; L3 is a linker peptide connecting the variable domains of the scFv; L4 is a linker peptide connecting the antibody of the first specificity to the antibody of the second specificity; CL is the light-chain constant domain; and CH1, CH2, CH3 are the heavy chain constant domains. The first and second specificities can be toward any antigen or epitope.

In some embodiments of a T-cell engaging multispecific activatable antibody, one antigen is PD-1, and another antigen is typically a stimulatory (also referred to herein as activating) or inhibitory receptor present on the surface of a T-cell, natural killer (NK) cell, myeloid mononuclear cell, macrophage, and/or other immune effector cell, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137 (also referred to as TNFRSF9), CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. The antibody domain conferring specificity to the T-cell surface antigen can also be substituted by a ligand or ligand domain that binds to a T-cell receptor, a NK-cell receptor, a macrophage receptor, and/or other immune effector cell receptor.

In some embodiments, the targeting antibody is an anti-PD-1 antibody disclosed herein. In some embodiments, the targeting antibody can be in the form an activatable antibody. In some embodiments, the scFv(s) can be in the form of a Pro-scFv (see, e.g., WO 2009/025846, WO 2010/081173).

In some embodiments, the scFv is specific for binding CDR, and comprises or is derived from an antibody or fragment thereof that binds CD3ε, e.g., CH2527, FN18, H2C, OKT3, 2C11, UCHT1, or V9. In some embodiments, the scFv is specific for binding CTLA-4 (also referred to herein as CTLA and CTLA4).

In some embodiments, the anti-CTLA-4 scFv includes the amino acid sequence:

(SEQ ID NO: 585) GGGSGGGGSGSGGGSGGGGSGGGEIVLTQSPGTLSLSPGERATLSCRASQ SVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRSGGSTITSYNVYYTKLSS SGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEW VSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA TNSLYWYFDLWGRGTLVTVSSAS

In some embodiments, the anti-CTLA-4 scFv includes the amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 585.

In some embodiments, the anti-CDR scFv includes the amino acid sequence:

(SEQ ID NO: 586) GGGSGGGGSGSGGGSGGGGSGGGQVQLQQSGAELARPGASVKMSCKASGY TFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSS TAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSG GGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKS GTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQ QWSSNPFTFGSGTKLEINR

In some embodiments, the anti-CDR scFv includes the amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 586.

In some embodiments, the scFv is specific for binding one or more T-cells, one or more NK-cells and/or one or more macrophages. In some embodiments, the scFv is specific for binding a target selected from the group consisting of B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, TIGIT, TIM3, or VISTA.

In some embodiments, the multispecific activatable antibody also includes an agent conjugated to the AB. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is conjugated to the multispecific activatable antibody via a linker. In some embodiments, the agent is conjugated to the AB via a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

In some embodiments, the multispecific activatable antibody also includes a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

In some embodiments, the multispecific activatable antibody naturally contains one or more disulfide bonds. In some embodiments, the multispecific activatable antibody can be engineered to include one or more disulfide bonds.

The disclosure also provides an isolated nucleic acid molecule encoding a multispecific activatable antibody described herein, as well as vectors that include these isolated nucleic acid sequences. The disclosure provides methods of producing a multispecific activatable antibody by culturing a cell under conditions that lead to expression of the activatable antibody, wherein the cell comprises such a nucleic acid molecule. In some embodiments, the cell comprises such a vector.

The disclosure also provides a method of manufacturing multispecific activatable antibodies of the disclosure by (a) culturing a cell comprising a nucleic acid construct that encodes the multispecific activatable antibody under conditions that lead to expression of the multispecific activatable, and (b) recovering the multispecific activatable antibody. Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein.

The disclosure also provides multispecific activatable antibodies and/or multispecific activatable antibody compositions that include at least a first antibody or antigen-binding fragment thereof (AB1) that specifically binds a first target or first epitope and a second antibody or antigen-biding fragment thereof (AB2) that binds a second target or a second epitope, where at least AB1 is coupled or otherwise attached to a masking moiety (MM1), such that coupling of the MM1 reduces the ability of AB1 to bind its target. In some embodiments, the MM1 is coupled to AB1 via a first cleavable moiety (CM1) sequence that includes a substrate for a protease, for example, a protease that is co-localized with the target of AB1 at a treatment site or a diagnostic site in a subject. The multispecific activatable antibodies provided herein are stable in circulation, activated at intended sites of therapy and/or diagnosis but not in normal, i.e., healthy tissue, and, when activated, exhibit binding to the target of AB1 that is at least comparable to the corresponding, unmodified multispecific antibody. Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein.

The disclosure also provides compositions and methods that include a multispecific activatable antibody that includes at least a first antibody or antibody fragment (AB1) that specifically binds a target and a second antibody or antibody fragment (AB2), where at least the first AB in the multispecific activatable antibody is coupled to a masking moiety (MM1) that decreases the ability of AB1 to bind its target. In some embodiments, each AB is coupled to a MM that decreases the ability of its corresponding AB to each target. For example, in bispecific activatable antibody embodiments, AB1 is coupled to a first masking moiety (MM1) that decreases the ability of AB1 to bind its target, and AB2 is coupled to a second masking moiety (MM2) that decreases the ability of AB2 to bind its target. In some embodiments, the multispecific activatable antibody comprises more than two AB regions; in such embodiments, AB1 is coupled to a first masking moiety (MM1) that decreases the ability of AB1 to bind its target, AB2 is coupled to a second masking moiety (MM2) that decreases the ability of AB2 to bind its target, AB3 is coupled to a third masking moiety (MM3) that decreases the ability of AB3 to bind its target, and so on for each AB in the multispecific activatable antibody. Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein.

In some embodiments, the multispecific activatable antibody further includes at least one cleavable moiety (CM) that is a substrate for a protease, where the CM links a MM to an AB. For example, in some embodiments, the multispecific activatable antibody includes at least a first antibody or antibody fragment (AB1) that specifically binds a target and a second antibody or antibody fragment (AB2), where at least the first AB in the multispecific activatable antibody is coupled via a first cleavable moiety (CM1) to a masking moiety (MM1) that decreases the ability of AB1 to bind its target. In some bispecific activatable antibody embodiments, AB1 is coupled via CM1 to MM1, and AB2 is coupled via a second cleavable moiety (CM2) to a second masking moiety (MM2) that decreases the ability of AB2 to bind its target. In some embodiments, the multispecific activatable antibody comprises more than two AB regions; in some of these embodiments, AB1 is coupled via CM1 to MM1, AB2 is coupled via CM2 to MM2, and AB3 is coupled via a third cleavable moiety (CM3) to a third masking moiety (MM3) that decreases the ability of AB3 to bind its target, and so on for each AB in the multispecific activatable antibody. Suitable AB, MM, and/or CM include any of the AB, MM, and/or CM disclosed herein.

Activatable Antibodies Having Non-Binding Steric Moieties or Binding Partners for Non-Binding Steric Moieties

The disclosure also provides activatable antibodies that include non-binding steric moieties (NB) or binding partners (BP) for non-binding steric moieties, where the BP recruits or otherwise attracts the NB to the activatable antibody. The activatable antibodies provided herein include, for example, an activatable antibody that includes a non-binding steric moiety (NB), a cleavable linker (CL) and antibody or antibody fragment (AB) that binds a target; an activatable antibody that includes a binding partner for a non-binding steric moiety (BP), a CL and an AB; and an activatable antibody that includes a BP to which an NB has been recruited, a CL and an AB that binds the target. Activatable antibodies in which the NB is covalently linked to the CL and AB of the activatable antibody or is associated by interaction with a BP that is covalently linked to the CL and AB of the activatable antibody are referred to herein as “NB-containing activatable antibodies.” By activatable or switchable is meant that the activatable antibody exhibits a first level of binding to a target when the activatable antibody is in an inhibited, masked or uncleaved state (i.e., a first conformation), and a second level of binding to the target when the activatable antibody is in an uninhibited, unmasked and/or cleaved state (i.e., a second conformation, i.e., activated antibody), where the second level of target binding is greater than the first level of target binding. The activatable antibody compositions can exhibit increased bioavailability and more favorable biodistribution compared to conventional antibody therapeutics.

In some embodiments, activatable antibodies provide for reduced toxicity and/or adverse side effects that could otherwise result from binding of the at non-treatment sites and/or non-diagnostic sites if the AB were not masked or otherwise inhibited from binding to such a site.

Anti-PD-1 activatable antibodies that include a non-binding steric moiety (NB) can be made using the methods set forth in PCT Publication No. WO 2013/192546, the contents of which are hereby incorporated by reference in their entirety.

Use of Antibodies and Activatable Antibodies

It will be appreciated that administration of therapeutic entities in accordance with the disclosure will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures can be appropriate in treatments and therapies in accordance with the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman W N “Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists.

Therapeutic formulations of the disclosure, which include an anti-PD-1 antibody and/or activatable anti-PD-1 antibody, such as by way of non-limiting example, an antibody and/or an activatable antibody, are used to prevent, treat or otherwise ameliorate a disease or disorder associated with aberrant target expression and/or activity. For example, therapeutic formulations of the disclosure, which include an antibody and/or an activatable antibody, are used to treat or otherwise ameliorate a cancer or other neoplastic condition, inflammation, an inflammatory disorder, and/or an autoimmune disease. In some embodiments, the cancer is a solid tumor or a hematologic malignancy where the target is expressed. In some embodiments, the cancer is a solid tumor where the target is expressed. In some embodiments, the cancer is a hematologic malignancy where the target is expressed. In some embodiments, the target is expressed on parenchyma (e.g., in cancer, the portion of an organ or tissue that often carries out function(s) of the organ or tissue). In some embodiments, the target is expressed on a cell, tissue, or organ. In some embodiments, the target is expressed on stroma (i.e., the connective supportive framework of a cell, tissue, or organ). In some embodiments, the target is expressed on an osteoblast. In some embodiments, the target is expressed on the endothelium (vasculature). In some embodiments, the target is expressed on a cancer stem cell.

Efficaciousness of prevention, amelioration or treatment is determined in association with any known method for diagnosing or treating the disease or disorder associated with target expression and/or activity, such as, for example, aberrant target expression and/or activity. Prolonging the survival of a subject or otherwise delaying the progression of the disease or disorder associated with target expression and/or activity, e.g., aberrant target expression and/or activity, in a subject indicates that the antibody and/or activatable antibody confers a clinical benefit.

An antibody and/or an activatable antibody can be administered in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

In some embodiments where antibody fragments are used, the smallest fragment that specifically binds to the binding domain of the target protein is selected. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also contain more than one active compounds as necessary for the particular indication being treated, for example, in some embodiments, those with complementary activities that do not adversely affect each other. In some embodiments, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

In some embodiments, the antibody and/or activatable antibody contains a detectable label. An intact antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)₂) is used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the disclosure can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, immunochemical staining, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

The antibodies and/or activatable antibodies of the disclosure are also useful in a variety of diagnostic and prophylactic formulations. In one embodiment, an antibody and/or an activatable antibody is administered to patients that are at risk of developing one or more of the aforementioned disorders. A patient's or organ's predisposition to one or more of the aforementioned disorders can be determined using genotypic, serological or biochemical markers.

In some embodiments of the disclosure, an antibody and/or an activatable antibody is administered to human individuals diagnosed with a clinical indication associated with one or more of the aforementioned disorders. Upon diagnosis, an antibody and/or an activatable antibody is administered to mitigate or reverse the effects of the clinical indication.

An antibody and/or an activatable antibody of the disclosure is also useful in the detection of a target in patient samples and accordingly are useful as diagnostics. For example, the antibodies and/or activatable antibodies, and conjugated versions thereof, of the disclosure are used in in vitro assays, e.g., ELISA, to detect target levels in a patient sample.

In some embodiments, anti-PDL1 antibodies are used as a diagnostic for patients who are more likely to respond favorably to treatment with an anti-PD-1 antibody and/or activatable antibody of the disclosure. In these embodiments, PD-L1 expression in tumor biopsies on both tumor cells and infiltrating immune cells and PD-L1 expression on immune cells in blood are used to indicate the presence of active immunity and, thus, potential for suppression of T cell activity through the engagement of PD-1 by PD-L1.

In one embodiment, an antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody of the disclosure is immobilized on a solid support (e.g., the well(s) of a microtiter plate). The immobilized antibody, conjugated antibody, activatable antibody and/or conjugated activatable antibody serves as a capture antibody for any target that can be present in a test sample. Prior to contacting the immobilized antibody and/or activatable antibody, and/or conjugated versions thereof, with a patient sample, the solid support is rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte.

Subsequently the wells are treated with a test sample suspected of containing the antigen, or with a solution containing a standard amount of the antigen. Such a sample is, e.g., a serum sample from a subject suspected of having levels of circulating antigen considered to be diagnostic of a pathology. After rinsing away the test sample or standard, the solid support is treated with a second antibody that is detectably labeled. The labeled second antibody serves as a detecting antibody. The level of detectable label is measured, and the concentration of target antigen in the test sample is determined by comparison with a standard curve developed from the standard samples.

It will be appreciated that based on the results obtained using the antibodies and activatable antibodies of the disclosure, and conjugated versions thereof, in an in vitro diagnostic assay, it is possible to stage a disease in a subject based on expression levels of the target antigen. For a given disease, samples of blood are taken from subjects diagnosed as being at various stages in the progression of the disease, and/or at various points in the therapeutic treatment of the disease. Using a population of samples that provides statistically significant results for each stage of progression or therapy, a range of concentrations of the antigen that can be considered characteristic of each stage is designated.

An antibody, a conjugated antibody, an activatable antibody and/or a conjugated activatable antibody can also be used in diagnostic and/or imaging methods. In some embodiments, such methods are in vitro methods. In some embodiments, such methods are in vivo methods. In some embodiments, such methods are in situ methods. In some embodiments, such methods are ex vivo methods. For example, activatable antibodies having an enzymatically cleavable CM can be used to detect the presence or absence of an enzyme that is capable of cleaving the CM. Such activatable antibodies can be used in diagnostics, which can include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment of increased reduction potential such as that which can provide for reduction of a disulfide bond) through measured accumulation of activated antibodies (i.e., antibodies resulting from cleavage of an activatable antibody) in a given cell or tissue of a given host organism. Such accumulation of activated antibodies indicates not only that the tissue expresses enzymatic activity (or an increased reduction potential depending on the nature of the CM) but also that the tissue expresses target to which the activated antibody binds.

For example, the CM can be selected to be substrate for at least one protease found at the site of a tumor, at the site of a viral or bacterial infection at a biologically confined site (e.g., such as in an abscess, in an organ, and the like), and the like. The AB can be one that binds a target antigen. Using methods as disclosed herein, or when appropriate, methods familiar to one skilled in the art, a detectable label (e.g., a fluorescent label or radioactive label or radiotracer) can be conjugated to an AB or other region of an antibody and/or activatable antibody. Suitable detectable labels are discussed in the context of the above screening methods and additional specific examples are provided below. Using an AB specific to a protein or peptide of the disease state, along with at least one protease whose activity is elevated in the disease tissue of interest, activatable antibodies will exhibit an increased rate of binding to disease tissue relative to tissues where the CM specific enzyme is not present at a detectable level or is present at a lower level than in disease tissue or is inactive (e.g., in zymogen form or in complex with an inhibitor). Since small proteins and peptides are rapidly cleared from the blood by the renal filtration system, and because the enzyme specific for the CM is not present at a detectable level (or is present at lower levels in non-disease tissues or is present in inactive conformation), accumulation of activated antibodies in the disease tissue is enhanced relative to non-disease tissues.

In another example, activatable antibodies can be used to detect the presence or absence of a cleaving agent in a sample. For example, where the activatable antibodies contain a CM susceptible to cleavage by an enzyme, the activatable antibodies can be used to detect (either qualitatively or quantitatively) the presence of an enzyme in the sample. In another example, where the activatable antibodies contain a CM susceptible to cleavage by reducing agent, the activatable antibodies can be used to detect (either qualitatively or quantitatively) the presence of reducing conditions in a sample. To facilitate analysis in these methods, the activatable antibodies can be detectably labeled, and can be bound to a support (e.g., a solid support, such as a slide or bead). The detectable label can be positioned on a portion of the activatable antibody that is not released following cleavage, for example, the detectable label can be a quenched fluorescent label or other label that is not detectable until cleavage has occurred. The assay can be conducted by, for example, contacting the immobilized, detectably labeled activatable antibodies with a sample suspected of containing an enzyme and/or reducing agent for a time sufficient for cleavage to occur, then washing to remove excess sample and contaminants. The presence or absence of the cleaving agent (e.g., enzyme or reducing agent) in the sample is then assessed by a change in detectable signal of the activatable antibodies prior to contacting with the sample e.g., the presence of and/or an increase in detectable signal due to cleavage of the activatable antibody by the cleaving agent in the sample.

Such detection methods can be adapted to also provide for detection of the presence or absence of a target that is capable of binding the AB of the activatable antibodies when cleaved. Thus, the assays can be adapted to assess the presence or absence of a cleaving agent and the presence or absence of a target of interest. The presence or absence of the cleaving agent can be detected by the presence of and/or an increase in detectable label of the activatable antibodies as described above, and the presence or absence of the target can be detected by detection of a target-AB complex e.g., by use of a detectably labeled anti-target antibody.

Activatable antibodies are also useful in in situ imaging for the validation of activatable antibody activation, e.g., by protease cleavage, and binding to a particular target. In situ imaging is a technique that enables localization of proteolytic activity and target in biological samples such as cell cultures or tissue sections. Using this technique, it is possible to confirm both binding to a given target and proteolytic activity based on the presence of a detectable label (e.g., a fluorescent label).

These techniques are useful with any frozen cells or tissue derived from a disease site (e.g. tumor tissue) or healthy tissues. These techniques are also useful with fresh cell or tissue samples.

In these techniques, an activatable antibody is labeled with a detectable label. The detectable label can be a fluorescent dye, (e.g. a fluorophore, Fluorescein Isothiocyanate (FITC), Rhodamine Isothiocyanate (TRITC), an Alexa Fluor® label), a near infrared (NIR) dye (e.g., Qdot® nanocrystals), a colloidal metal, a hapten, a radioactive marker, biotin and an amplification reagent such as streptavidin, or an enzyme (e.g. horseradish peroxidase or alkaline phosphatase).

Detection of the label in a sample that has been incubated with the labeled, activatable antibody indicates that the sample contains the target and contains a protease that is specific for the CM of the activatable antibody. In some embodiments, the presence of the protease can be confirmed using broad spectrum protease inhibitors such as those described herein, and/or by using an agent that is specific for the protease, for example, an antibody such as A11, which is specific for the protease matriptase and inhibits the proteolytic activity of matriptase; see e.g., International Publication Number WO 2010/129609, published 11 Nov. 2010. The same approach of using broad spectrum protease inhibitors such as those described herein, and/or by using a more selective inhibitory agent can be used to identify a protease that is specific for the CM of the activatable antibody. In some embodiments, the presence of the target can be confirmed using an agent that is specific for the target, e.g., another antibody, or the detectable label can be competed with unlabeled target. In some embodiments, unlabeled activatable antibody could be used, with detection by a labeled secondary antibody or more complex detection system.

Similar techniques are also useful for in vivo imaging where detection of the fluorescent signal in a subject, e.g., a mammal, including a human, indicates that the disease site contains the target and contains a protease that is specific for the CM of the activatable antibody.

These techniques are also useful in kits and/or as reagents for the detection, identification or characterization of protease activity in a variety of cells, tissues, and organisms based on the protease-specific CM in the activatable antibody.

The disclosure provides methods of using the antibodies and/or activatable antibodies in a variety of diagnostic and/or prophylactic indications. For example, the disclosure provides methods of detecting presence or absence of a cleaving agent and a target of interest in a subject or a sample by (i) contacting a subject or sample with an activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target of interest, wherein the activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form of a natural binding partner of the AB; and (b) wherein, in an uncleaved, non-activated state, the MM interferes with specific binding of the AB to the target, and in a cleaved, activated state the MM does not interfere or compete with specific binding of the AB to the target; and (ii) measuring a level of activated activatable antibody in the subject or sample, wherein a detectable level of activated activatable antibody in the subject or sample indicates that the cleaving agent and the target are present in the subject or sample and wherein no detectable level of activated activatable antibody in the subject or sample indicates that the cleaving agent, the target or both the cleaving agent and the target are absent and/or not sufficiently present in the subject or sample. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.

The disclosure also provides methods of detecting presence or absence of a cleaving agent in a subject or a sample by (i) contacting a subject or sample with an activatable antibody in the presence of a target of interest, e.g., the target, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target of interest, wherein the activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form of a natural binding partner of the AB; and (b) wherein, in an uncleaved, non-activated state, the MM interferes with specific binding of the AB to the target, and in a cleaved, activated state the MM does not interfere or compete with specific binding of the AB to the target; and (ii) measuring a level of activated activatable antibody in the subject or sample, wherein a detectable level of activated activatable antibody in the subject or sample indicates that the cleaving agent is present in the subject or sample and wherein no detectable level of activated activatable antibody in the subject or sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or sample. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.

The disclosure also provides kits for use in methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample, where the kits include at least an activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target of interest, wherein the activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form of a natural binding partner of the AB; and (b) wherein, in an uncleaved, non-activated state, the MM interferes with specific binding of the AB to the target, and in a cleaved, activated state the MM does not interfere or compete with specific binding of the AB to the target; and (ii) measuring a level of activated activatable antibody in the subject or sample, wherein a detectable level of activated activatable antibody in the subject or sample indicates that the cleaving agent is present in the subject or sample and wherein no detectable level of activated activatable antibody in the subject or sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or sample. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.

The disclosure also provides methods of detecting presence or absence of a cleaving agent in a subject or a sample by (i) contacting a subject or sample with an activatable antibody, wherein the activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, an antigen binding domain (AB) that specifically binds the target, and a detectable label, wherein the activatable antibody in an uncleaved, non-activated state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB and is not a modified form of a natural binding partner of the AB; wherein, in an uncleaved, non-activated state, the MM interferes with specific binding of the AB to the target, and in a cleaved, activated state the MM does not interfere or compete with specific binding of the AB to the target; and wherein the detectable label is positioned on a portion of the activatable antibody that is released following cleavage of the CM; and (ii) measuring a level of detectable label in the subject or sample, wherein a detectable level of the detectable label in the subject or sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or sample and wherein no detectable level of the detectable label in the subject or sample indicates that the cleaving agent is present in the subject or sample. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.

The disclosure also provides kits for use in methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample, where the kits include at least an activatable antibody and/or conjugated activatable antibody (e.g., an activatable antibody to which a therapeutic agent is conjugated) described herein for use in contacting a subject or biological sample and means for detecting the level of activated activatable antibody and/or conjugated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent and the target are present in the subject or biological sample and wherein no detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent, the target or both the cleaving agent and the target are absent and/or not sufficiently present in the subject or biological sample, such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample.

The disclosure also provides methods of detecting presence or absence of a cleaving agent in a subject or a sample by (i) contacting a subject or biological sample with an activatable antibody in the presence of the target, and (ii) measuring a level of activated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is present in the subject or biological sample and wherein no detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or biological sample at a detectable level, such that protease cleavage of the activatable antibody cannot be detected in the subject or biological sample. Such an activatable antibody includes a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target, wherein the activatable antibody in an uncleaved (i.e., non-activated) state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB; and (b) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an activatable antibody in a cleaved (i.e., activated) state does not interfere or compete with specific binding of the AB to the target. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the detectable label is attached to the masking moiety. In some embodiments, the detectable label is attached to the cleavable moiety N-terminal to the protease cleavage site. In some embodiments, a single antigen binding site of the AB is masked. In some embodiments wherein an antibody of the disclosure has at least two antigen binding sites, at least one antigen binding site is masked and at least one antigen binding site is not masked. In some embodiments all antigen binding sites are masked. In some embodiments, the measuring step includes use of a secondary reagent comprising a detectable label.

The disclosure also provides kits for use in methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample, where the kits include at least an activatable antibody and/or conjugated activatable antibody described herein for use in contacting a subject or biological sample with an activatable antibody in the presence of the target, and measuring a level of activated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is present in the subject or biological sample and wherein no detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or biological sample at a detectable level, such that protease cleavage of the activatable antibody cannot be detected in the subject or biological sample. Such an activatable antibody includes a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, e.g., a protease, and an antigen binding domain or fragment thereof (AB) that specifically binds the target, wherein the activatable antibody in an uncleaved (i.e., non-activated) state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB; and (b) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an activatable antibody in a cleaved (i.e., activated) state does not interfere or compete with specific binding of the AB to the target. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the detectable label is attached to the masking moiety. In some embodiments, the detectable label is attached to the cleavable moiety N-terminal to the protease cleavage site. In some embodiments, a single antigen binding site of the AB is masked. In some embodiments wherein an antibody of the disclosure has at least two antigen binding sites, at least one antigen binding site is masked and at least one antigen binding site is not masked. In some embodiments all antigen binding sites are masked. In some embodiments, the measuring step includes use of a secondary reagent comprising a detectable label.

The disclosure also provides kits for use in methods of detecting presence or absence of a cleaving agent in a subject or a sample, where the kits include at least an activatable antibody and/or conjugated activatable antibody described herein for use in contacting a subject or biological sample and means for detecting the level of activated activatable antibody and/or conjugated activatable antibody in the subject or biological sample, wherein the activatable antibody includes a detectable label that is positioned on a portion of the activatable antibody that is released following cleavage of the CM, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or biological sample such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein no detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent is present in the subject or biological sample at a detectable level.

The disclosure provides methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample by (i) contacting a subject or biological sample with an activatable antibody, wherein the activatable antibody includes a detectable label that is positioned on a portion of the activatable antibody that is released following cleavage of the CM and (ii) measuring a level of activated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent, the target or both the cleaving agent and the target are absent and/or not sufficiently present in the subject or biological sample, such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein a reduced detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent and the target are present in the subject or biological sample. A reduced level of detectable label is, for example, a reduction of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%. Such an activatable antibody includes a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, and an antigen binding domain or fragment thereof (AB) that specifically binds the target, wherein the activatable antibody in an uncleaved (i.e., non-activated) state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB; and (b) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an activatable antibody in a cleaved (i.e., activated) state does not interfere or compete with specific binding of the AB to the target. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.

The disclosure also provides kits for use in methods of detecting presence or absence of a cleaving agent and the target in a subject or a sample, where the kits include at least an activatable antibody and/or conjugated activatable antibody described herein for use in contacting a subject or biological sample and means for detecting the level of activated activatable antibody and/or conjugated activatable antibody in the subject or biological sample, wherein a detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent, the target or both the cleaving agent and the target are absent and/or not sufficiently present in the subject or biological sample, such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein a reduced detectable level of activated activatable antibody in the subject or biological sample indicates that the cleaving agent and the target are present in the subject or biological sample. A reduced level of detectable label is, for example, a reduction of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%.

The disclosure also provides methods of detecting presence or absence of a cleaving agent in a subject or a sample by (i) contacting a subject or biological sample with an activatable antibody, wherein the activatable antibody includes a detectable label that is positioned on a portion of the activatable antibody that is released following cleavage of the CM; and (ii) measuring a level of detectable label in the subject or biological sample, wherein a detectable level of the detectable label in the subject or biological sample indicates that the cleaving agent is absent and/or not sufficiently present in the subject or biological sample at a detectable level, such that protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein a reduced detectable level of the detectable label in the subject or biological sample indicates that the cleaving agent is present in the subject or biological sample. A reduced level of detectable label is, for example, a reduction of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%. Such an activatable antibody includes a masking moiety (MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, and an antigen binding domain or fragment thereof (AB) that specifically binds the target, wherein the activatable antibody in an uncleaved (i.e., non-activated) state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of the AB to the target, and wherein the MM does not have an amino acid sequence of a naturally occurring binding partner of the AB; and (b) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to the target, and wherein the MM of an activatable antibody in a cleaved (i.e., activated) state does not interfere or compete with specific binding of the AB to the target. In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.

The disclosure also provides kits for use in methods of detecting presence or absence of a cleaving agent of interest in a subject or a sample, where the kits include at least an activatable antibody and/or conjugated activatable antibody described herein for use in contacting a subject or biological sample and means for detecting the level of activated activatable antibody and/or conjugated activatable antibody in the subject or biological sample, wherein the activatable antibody includes a detectable label that is positioned on a portion of the activatable antibody that is released following cleavage of the CM, wherein a detectable level of the detectable label in the subject or biological sample indicates that the cleaving agent, the target, or both the cleaving agent and the target are absent and/or not sufficiently present in the subject or biological sample, such that the target binding and/or protease cleavage of the activatable antibody cannot be detected in the subject or biological sample, and wherein a reduced detectable level of the detectable label in the subject or biological sample indicates that the cleaving agent and the target are present in the subject or biological sample. A reduced level of detectable label is, for example, a reduction of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about 100%.

In some embodiments of these methods and kits, the activatable antibody includes a detectable label. In some embodiments of these methods and kits, the detectable label includes an imaging agent, a contrasting agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments of these methods and kits, the imaging agent comprises a radioisotope. In some embodiments of these methods and kits, the radioisotope is indium or technetium. In some embodiments of these methods and kits, the contrasting agent comprises iodine, gadolinium or iron oxide. In some embodiments of these methods and kits, the enzyme comprises horseradish peroxidase, alkaline phosphatase, or (3-galactosidase. In some embodiments of these methods and kits, the fluorescent label comprises yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In some embodiments of these methods and kits, the luminescent label comprises an N-methylacrydium derivative. In some embodiments of these methods, the label comprises an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments of these methods and kits, the ligand-based label comprises biotin, avidin, streptavidin or one or more haptens.

In some embodiments of these methods and kits, the subject is a mammal. In some embodiments of these methods and kits, the subject is a human. In some embodiments, the subject is a non-human mammal, such as a non-human primate, companion animal (e.g., cat, dog, horse), farm animal, work animal, or zoo animal. In some embodiments, the subject is a rodent.

In some embodiments of these methods, the method is an in vivo method. In some embodiments of these methods, the method is an in situ method. In some embodiments of these methods, the method is an ex vivo method. In some embodiments of these methods, the method is an in vitro method.

In some embodiments, in situ imaging and/or in vivo imaging are useful in methods to identify which patients to treat. For example, in in situ imaging, the activatable antibodies are used to screen patient samples to identify those patients having the appropriate protease(s) and target(s) at the appropriate location, e.g., at a tumor site.

In some embodiments in situ imaging is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure. For example, patients that test positive for both the target (e.g., the target) and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM. Likewise, patients that test negative for either or both of the target (e.g., the target) and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods might be identified as suitable candidates for another form of therapy. In some embodiments, such patients that test negative with respect to a first activatable antibody can be tested with other activatable antibodies comprising different CMs until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease). In some embodiments, the patient is then administered a therapeutically effective amount of the activatable antibody for which the patient tested positive.

In some embodiments in vivo imaging is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure. For example, patients that test positive for both the target (e.g., the target) and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM. Likewise, patients that test negative might be identified as suitable candidates for another form of therapy. In some embodiments, such patients that test negative with respect to a first activatable antibody can be tested with other activatable antibodies comprising different CMs until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease). In some embodiments, the patient is then administered a therapeutically effective amount of the activatable antibody for which the patient tested positive.

In some embodiments of the methods and kits, the method or kit is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure. For example, patients that test positive for both the target (e.g., the target) and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested in these methods are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM. Likewise, patients that test negative for both of the targets (e.g., the target) and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods might be identified as suitable candidates for another form of therapy. In some embodiments, such patients can be tested with other activatable antibodies until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease). In some embodiments, patients that test negative for either of the target (e.g., the target) are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM. In some embodiments, patients that test negative for either of the target (e.g., the target) are identified as not being suitable candidates for treatment with such an activatable antibody comprising such a CM. In some embodiments, such patients can be tested with other activatable antibodies until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease). In some embodiments, the activatable antibody is an activatable antibody to which a therapeutic agent is conjugated. In some embodiments, the activatable antibody is not conjugated to an agent. In some embodiments, the activatable antibody comprises a detectable label. In some embodiments, the detectable label is positioned on the AB. In some embodiments, measuring the level of activatable antibody in the subject or sample is accomplished using a secondary reagent that specifically binds to the activated antibody, wherein the reagent comprises a detectable label. In some embodiments, the secondary reagent is an antibody comprising a detectable label.

In some embodiments, a method or kit is used to identify or otherwise refine a patient population suitable for treatment with an anti-the target activatable antibody and/or conjugated activatable antibody (e.g., activatable antibody to which a therapeutic agent is conjugated) of the disclosure, followed by treatment by administering that activatable antibody and/or conjugated activatable antibody to a subject in need thereof. For example, patients that test positive for both the targets (e.g., the target) and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody and/or conjugated activatable antibody being tested in these methods are identified as suitable candidates for treatment with such antibody and/or such a conjugated activatable antibody comprising such a CM, and the patient is then administered a therapeutically effective amount of the activatable antibody and/or conjugated activatable antibody that was tested. Likewise, patients that test negative for either or both of the target (e.g., the target) and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods might be identified as suitable candidates for another form of therapy. In some embodiments, such patients can be tested with other antibody and/or conjugated activatable antibody until a suitable antibody and/or conjugated activatable antibody for treatment is identified (e.g., an activatable antibody and/or conjugated activatable antibody comprising a CM that is cleaved by the patient at the site of disease). In some embodiments, the patient is then administered a therapeutically effective amount of the activatable antibody and/or conjugated activatable antibody for which the patient tested positive.

In some embodiments of these methods and kits, the MM is a peptide having a length from about 4 to 40 amino acids. In some embodiments of these methods and kits, the activatable antibody comprises a linker peptide, wherein the linker peptide is positioned between the MM and the CM. In some embodiments of these methods and kits, the activatable antibody comprises a linker peptide, where the linker peptide is positioned between the AB and the CM. In some embodiments of these methods and kits, the activatable antibody comprises a first linker peptide (L1) and a second linker peptide (L2), wherein the first linker peptide is positioned between the MM and the CM and the second linker peptide is positioned between the AB and the CM. In some embodiments of these methods and kits, each of L1 and L2 is a peptide of about 1 to 20 amino acids in length, and wherein each of L1 and L2 need not be the same linker. In some embodiments of these methods and kits, one or both of L1 and L2 comprises a glycine-serine polymer. In some embodiments of these methods and kits, at least one of L1 and L2 comprises an amino acid sequence selected from the group consisting of (GS)_(n), (GSGGS)_(n) (SEQ ID NO: 363) and (GGGS)_(n) (SEQ ID NO: 364), where n is an integer of at least one. In some embodiments of these methods and kits, at least one of L1 and L2 comprises an amino acid sequence having the formula (GGS)_(n), where n is an integer of at least one. In some embodiments of these methods and kits, at least one of L1 and L2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 365), GGSGG (SEQ ID NO: 366), GSGSG (SEQ ID NO: 367), GSGGG (SEQ ID NO: 368), GGGSG (SEQ ID NO: 369), and GSSSG (SEQ ID NO: 370).

In some embodiments of these methods and kits, the AB comprises an antibody or antibody fragment sequence selected from the cross-reactive antibody sequences presented herein. In some embodiments of these methods and kits, the AB comprises a Fab fragment, a scFv or a single chain antibody (scAb).

In some embodiments of these methods and kits, the cleaving agent is a protease that is co-localized in the subject or sample with the target and the CM is a polypeptide that functions as a substrate for the protease, wherein the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease. In some embodiments of these methods and kits, the CM is a polypeptide of up to 15 amino acids in length. In some embodiments of these methods and kits, the CM is coupled to the N-terminus of the AB. In some embodiments of these methods and kits, the CM is coupled to the C-terminus of the AB. In some embodiments of these methods and kits, the CM is coupled to the N-terminus of a VL chain of the AB.

The antibodies and/or activatable antibodies of the disclosure are used in diagnostic and prophylactic formulations. In one embodiment, an activatable antibody is administered to patients that are at risk of developing one or more of the aforementioned inflammation, inflammatory disorders, cancer or other disorders.

A patient's or organ's predisposition to one or more of the aforementioned disorders can be determined using genotypic, serological or biochemical markers.

In some embodiments of the disclosure, an antibody and/or an activatable antibody is administered to human individuals diagnosed with a clinical indication associated with one or more of the aforementioned disorders. Upon diagnosis, an antibody and/or an activatable antibody is administered to mitigate or reverse the effects of the clinical indication.

Antibodies and/or activatable antibodies of the disclosure are also useful in the detection of the target in patient samples and accordingly are useful as diagnostics. For example, the antibodies and/or activatable antibodies of the disclosure are used in in vitro assays, e.g., ELISA, to detect target levels in a patient sample.

In some embodiments, anti-PDL1 antibodies are used as a diagnostic for patients who are more likely to respond favorably to treatment with an anti-PD-1 antibody and/or activatable antibody of the disclosure. In these embodiments, PD-L1 expression in tumor biopsies on both tumor cells and infiltrating immune cells and PD-L1 expression on immune cells in blood are used to indicate the presence of active immunity and, thus, potential for suppression of T cell activity through the engagement of PD-1 by PD-L1.

In one embodiment, an antibody and/or activatable antibody of the disclosure is immobilized on a solid support (e.g., the well(s) of a microtiter plate). The immobilized antibody and/or activatable antibody serves as a capture antibody for any target that can be present in a test sample. Prior to contacting the immobilized antibody and/or activatable antibody with a patient sample, the solid support is rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte.

Subsequently the wells are treated with a test sample suspected of containing the antigen, or with a solution containing a standard amount of the antigen. Such a sample is, e.g., a serum sample from a subject suspected of having levels of circulating antigen considered to be diagnostic of a pathology. After rinsing away the test sample or standard, the solid support is treated with a second antibody that is detectably labeled. The labeled second antibody serves as a detecting antibody. The level of detectable label is measured, and the concentration of target antigen in the test sample is determined by comparison with a standard curve developed from the standard samples.

It will be appreciated that based on the results obtained using the antibodies and/or activatable antibodies of the disclosure in an in vitro diagnostic assay, it is possible to stage a disease in a subject based on expression levels of the target antigen. For a given disease, samples of blood are taken from subjects diagnosed as being at various stages in the progression of the disease, and/or at various points in the therapeutic treatment of the disease. Using a population of samples that provides statistically significant results for each stage of progression or therapy, a range of concentrations of the antigen that can be considered characteristic of each stage is designated.

Antibodies, conjugated antibodies, activatable antibodies and/or conjugated activatable antibodies can also be used in diagnostic and/or imaging methods. In some embodiments, such methods are in vitro methods. In some embodiments, such methods are in vivo methods. In some embodiments, such methods are in situ methods. In some embodiments, such methods are ex vivo methods. For example, activatable antibodies having an enzymatically cleavable CM can be used to detect the presence or absence of an enzyme that is capable of cleaving the CM. Such activatable antibodies can be used in diagnostics, which can include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment of increased reduction potential such as that which can provide for reduction of a disulfide bond) through measured accumulation of activated antibodies (i.e., antibodies resulting from cleavage of an activatable antibody) in a given cell or tissue of a given host organism. Such accumulation of activated antibodies indicates not only that the tissue expresses enzymatic activity (or an increased reduction potential depending on the nature of the CM) but also that the tissue expresses target to which the activated antibody binds.

For example, the CM can be selected to be a protease substrate for a protease found at the site of a tumor, at the site of a viral or bacterial infection at a biologically confined site (e.g., such as in an abscess, in an organ, and the like), and the like. The AB can be one that binds a target antigen. Using methods familiar to one skilled in the art, a detectable label (e.g., a fluorescent label or radioactive label or radiotracer) can be conjugated to an AB or other region of an activatable antibody. Suitable detectable labels are discussed in the context of the above screening methods and additional specific examples are provided below. Using an AB specific to a protein or peptide of the disease state, along with a protease whose activity is elevated in the disease tissue of interest, activatable antibodies will exhibit an increased rate of binding to disease tissue relative to tissues where the CM specific enzyme is not present at a detectable level or is present at a lower level than in disease tissue or is inactive (e.g., in zymogen form or in complex with an inhibitor). Since small proteins and peptides are rapidly cleared from the blood by the renal filtration system, and because the enzyme specific for the CM is not present at a detectable level (or is present at lower levels in non-disease tissues or is present in inactive conformation), accumulation of activated antibodies in the disease tissue is enhanced relative to non-disease tissues.

In another example, activatable antibodies can be used to detect the presence or absence of a cleaving agent in a sample. For example, where the activatable antibodies contain a CM susceptible to cleavage by an enzyme, the activatable antibodies can be used to detect (either qualitatively or quantitatively) the presence of an enzyme in the sample. In another example, where the activatable antibodies contain a CM susceptible to cleavage by reducing agent, the activatable antibodies can be used to detect (either qualitatively or quantitatively) the presence of reducing conditions in a sample. To facilitate analysis in these methods, the activatable antibodies can be detectably labeled, and can be bound to a support (e.g., a solid support, such as a slide or bead). The detectable label can be positioned on a portion of the activatable antibody that is not released following cleavage, for example, the detectable label can be a quenched fluorescent label or other label that is not detectable until cleavage has occurred. The assay can be conducted by, for example, contacting the immobilized, detectably labeled activatable antibodies with a sample suspected of containing an enzyme and/or reducing agent for a time sufficient for cleavage to occur, then washing to remove excess sample and contaminants. The presence or absence of the cleaving agent (e.g., enzyme or reducing agent) in the sample is then assessed by a change in detectable signal of the activatable antibodies prior to contacting with the sample e.g., the presence of and/or an increase in detectable signal due to cleavage of the activatable antibody by the cleaving agent in the sample.

Such detection methods can be adapted to also provide for detection of the presence or absence of a target that is capable of binding the AB of the activatable antibodies when cleaved. Thus, the assays can be adapted to assess the presence or absence of a cleaving agent and the presence or absence of a target of interest. The presence or absence of the cleaving agent can be detected by the presence of and/or an increase in detectable label of the activatable antibodies as described above, and the presence or absence of the target can be detected by detection of a target-AB complex e.g., by use of a detectably labeled anti-target antibody.

Activatable antibodies are also useful in in situ imaging for the validation of activatable antibody activation, e.g., by protease cleavage, and binding to a particular target. In situ imaging is a technique that enables localization of proteolytic activity and target in biological samples such as cell cultures or tissue sections. Using this technique, it is possible to confirm both binding to a given target and proteolytic activity based on the presence of a detectable label (e.g., a fluorescent label).

These techniques are useful with any frozen cells or tissue derived from a disease site (e.g. tumor tissue) or healthy tissues. These techniques are also useful with fresh cell or tissue samples.

In these techniques, an activatable antibody is labeled with a detectable label. The detectable label can be a fluorescent dye, (e.g. Fluorescein Isothiocyanate (FITC), Rhodamine Isothiocyanate (TRITC), a near infrared (NIR) dye (e.g., Qdot® nanocrystals), a colloidal metal, a hapten, a radioactive marker, biotin and an amplification reagent such as streptavidin, or an enzyme (e.g. horseradish peroxidase or alkaline phosphatase).

Detection of the label in a sample that has been incubated with the labeled, activatable antibody indicates that the sample contains the target and contains a protease that is specific for the CM of the activatable antibody. In some embodiments, the presence of the protease can be confirmed using broad spectrum protease inhibitors such as those described herein, and/or by using an agent that is specific for the protease, for example, an antibody such as A11, which is specific for the protease matriptase and inhibits the proteolytic activity of matriptase; see e.g., International Publication Number WO 2010/129609, published 11 Nov. 2010. The same approach of using broad spectrum protease inhibitors such as those described herein, and/or by using a more selective inhibitory agent can be used to identify a protease or class of proteases specific for the CM of the activatable antibody. In some embodiments, the presence of the target can be confirmed using an agent that is specific for the target, e.g., another antibody, or the detectable label can be competed with unlabeled target. In some embodiments, unlabeled activatable antibody could be used, with detection by a labeled secondary antibody or more complex detection system.

Similar techniques are also useful for in vivo imaging where detection of the fluorescent signal in a subject, e.g., a mammal, including a human, indicates that the disease site contains the target and contains a protease that is specific for the CM of the activatable antibody.

These techniques are also useful in kits and/or as reagents for the detection, identification or characterization of protease activity in a variety of cells, tissues, and organisms based on the protease-specific CM in the activatable antibody.

In some embodiments, in situ imaging and/or in vivo imaging are useful in methods to identify which patients to treat. For example, in in situ imaging, the activatable antibodies are used to screen patient samples to identify those patients having the appropriate protease(s) and target(s) at the appropriate location, e.g., at a tumor site.

In some embodiments in situ imaging is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure. For example, patients that test positive for both the target and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM. Likewise, patients that test negative for either or both of the target and the protease that cleaves the substrate in the CM in the activatable antibody being tested using these methods are identified as suitable candidates for another form of therapy (i.e., not suitable for treatment with the activatable antibody being tested). In some embodiments, such patients that test negative with respect to a first activatable antibody can be tested with other activatable antibodies comprising different CMs until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease).

In some embodiments in vivo imaging is used to identify or otherwise refine a patient population suitable for treatment with an activatable antibody of the disclosure. For example, patients that test positive for both the target and a protease that cleaves the substrate in the cleavable moiety (CM) of the activatable antibody being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such an activatable antibody comprising such a CM. Likewise, patients that test negative are identified as suitable candidates for another form of therapy (i.e., not suitable for treatment with the activatable antibody being tested). In some embodiments, such patients that test negative with respect to a first activatable antibody can be tested with other activatable antibodies comprising different CMs until a suitable activatable antibody for treatment is identified (e.g., an activatable antibody comprising a CM that is cleaved by the patient at the site of disease).

Pharmaceutical Compositions

The antibodies and/or activatable antibodies of the disclosure (also referred to herein as “active compounds”), and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody and/or activatable antibody and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES Example 1. Generation of Mouse Antibodies of the Embodiments that Bind Human PD-1 and Block hPD-L1 and hPD-L2 Binding to Human PD-1

This example demonstrates mouse antibodies of the disclosure that bind human PD-1 can be isolated from hybridomas derived from mouse immunization with recombinant human PD-1 protein, and that such binding can inhibit PD-1 binding to PDL1 and PDL2.

Six NZBWF1/J female mice (Jackson Laboratories, Sacramento, Calif.; cat #100008) were immunized in the right flank with recombinant human PD-1 (Sino Biological, Beijing, P.R. China; cat # ABIN2181605) on days 0, 7 and 21. Serum was taken from immunized mice on day 28 and binding to HEK293-hPD-1 (cells transfected with an expression vector encoding human PD-1 (Origene, cat # SC117011)) was measured. All six mice showed positive binding. Splenocytes were isolated from mouse 1, 3 and 6 and fused with SP0 mouse B-cells; likewise splenocytes were isolated from mouse 2, 4 and 5 and fused with SP0 mouse B-cells, giving rise to pools of hybridomas, m136 and m245. The hybridoma pools, m136 and m245, arising from the two fusions were plated for single clones, and clonal culture supernatants were assayed for antibodies capable of binding to HEK293-hPD-1, and not to un-transfected HEK293, cells. Hybridoma clones expressing anti-PD-1 antibodies were chosen for further analysis.

The sequences of the antibodies used in the studies presented herein are shown below:

m136-M13-MHC723 mIgG1/K

MHC723HC.1 Variable Heavy Chain   Region Amino Acid Sequence: (SEQ ID NO: 1) EVKLVESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQTPAKRLEWVAY ISNSGGNAHYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCTRED YGTSPFVYWGQGTLVTVSA MHC723HC.1 Variable Heavy Chain  Region Nucleic Acid Sequence:  (SEQ ID NO: 2) GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAACCTGGAGGGTC CCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGGCTATGCCA TGTCTTGGGTTCGCCAGACTCCGGCGAAGAGGCTGGAGTGGGTCGCATAC ATTAGTAATAGTGGTGGTAACGCCCACTATCCAGACAGTGTAAAGGGCCG ATTCACCATCTCCAGAGACAATGCCAAGAACACCCTATACCTGCAAATGA GCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTACAAGAGAGGAC TACGGTACTAGTCCTTTTGTTTACTGGGGCCAAGGGACTCTGGTCACTGT CTCTGCA MHC723LC.3 Variable Light Chain  Region Amino Acid Sequence:  (SEQ ID NO: 3) DIVLTQSPASLAVSLGQRTTISCRASESVDNYGISFMNWFQQKPGQPPKL LIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAVYFCQQSKDVPW TFGGGTKLEIR MHC723LC.3 Variable Light Chain  Region Nucleic Acid Sequence:  (SEQ ID NO: 4) GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTTGGGCA GAGGACCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGATAATTATGGCA TTAGTTTTATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTC CTCATCTATGCTGCATCCAACCAAGGATCCGGGGTCCCTGCCAGGTTTAG TGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGG AGGATGATACTGCAGTGTATTTCTGTCAGCAAAGTAAGGACGTTCCGTGG ACGTTCGGTGGAGGCACCAAGCTGGAAATCAGAC m136-M19-MHC725 mIgG2b/K

(SEQ ID NO: 5) MEIC725HC.2 Variable heavy chain region amino acid sequence: EVQLQQSGPELVKPGDSVKMSCKASGYTFTDYYMDWVKQSHGKSLEWIGYIYPKNGGSSYNQKF KGKATLTVDKSSSTAYMELHSLTSEDSAVYYCARKVVATDYWGQGTTLTVSS (SEQ ID NO: 6) MEIC725HC.2 Variable heavy chain region nucleic acid sequence: GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGATTCAGTGAAGATGTCCT GCAAGGCTTCTGGCTACACATTCACTGACTACTACATGGACTGGGTGAAGCAGAGCCATGGAAA GAGCCITGAGIGGATTGGATATATTTATCCTAAAAATGGIGGITCCAGCTACAATCAGAAGTTC AAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCACAGCC TGACATCTGAGGACTCTGCAGICTATTACTGTGCAAGAAAGGICGTAGCTACGGACTACTGGGG CCAAGGCACCACTCTCACAGTCTCCTCA  (SEQ ID NO: 7) MEIC725LC.2 Variable light chain region amino acid sequence: DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIFWASIRESGV PDRFTGSGSGTDFTLTISSVKAEDRAVYYCQQCDSYPWTFGGGTKLEIK  (SEQ ID NO: 8) MIIC725LC.2 Variable light chain region nucleic acid sequence: GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGA GCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCA GCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTTCTGGGCATCTATTAGGGAATCTGGGGTC CCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGG CTGAAGACCGGGCAGTTTATTACTGTCAGCAATGTGATAGCTATCCGTGGACGTTCGGTGGAGG CACCAAACTGGAAATCAAAC  m245-M3-MHC728 mIgG2a/K

(SEQ ID NO: 9) MIIC728HC.4 Variable heavy chain region amino acid sequence: EVKLVESGGGLVKPGGSLKLSCAASGFTFSNYAMSWVRQTPAKRLEWVAYISNGGGDTHYPDSL KGRFTVSRDNAKNTLYLQMSSLKSEDTAMYYCARENYGTSPFVYWGQGTLVTVSA (SEQ ID NO: 10) MIIC728HC.4 Variable heavy chain region nucleic acid sequence: GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAACCTGGAGGGTCCCTGAAACTCTCCT GTGCAGCCTCTGGATTCACTTTCAGTAACTATGCCATGTCTTGGGTTCGCCAGACTCCGGCGAA GAGGCTGGAGTGGGTCGCATACATTAGTAATGGTGGTGGTGACACCCACTATCCAGACAGTTTA AAGGGCCGATTCACCGTCTCCAGAGACAATGCCAAGAACACCCTGTACCTACAAATGAGCAGTC TGAAGTCTGAGGACACGGCCATGTATTACTGTGCAAGAGAAAACTACGGTACTAGTCCCTTTGT TTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 11) MIIC728LC.2Variable light chain region amino acid sequence: DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQGSGVPA RFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKDVPWTFGGGTKLEIK (SEQ ID NO: 12) MIIC728LC.2 Variable light chain region nucleic acid sequence: GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCT CCTGCAGAGCCAGCGAAAGTGTTGATAATTATGGCATTAGTTTTATGAACTGGTTCCAACAGAA ACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAACCAAGGATCCGGGGTCCCTGCC AGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATG ATACTGCAATGTATTTCTGTCAGCAAAGTAAAGATGTTCCGTGGACGTTCGGTGGAGGCACCAA GCTGGAAATCAAAC m245-M5-MHC729 mIgG1/K

(SEQ ID NO: 13) MEIC729HC.1 Variable heavy chain region amino acid sequence: EVQLVESGGGLVKSGGSLKLSCAHSGFSFSSYDMSWVRQTPAKRLEWVATISGGGRYTYYPDSV KGRFTISRDNAKNTLYLQMSGLRSEDTAMYYCASNYYGFDYWGQGTTLTVSS (SEQ ID NO: 14) MEIC729HC.1 Variable heavy chain region nucleic acid sequence: GAAGTGCAGCTGGTGGAGICIGGGGGAGGCTTAGTGAAGICTGGAGGGICCCTGAAACTCTCCT GTGCGCATTCTGGATTCAGTTTTAGTAGTTATGACATGTCTTGGGTTCGCCAGACTCCGGCGAA GAGGCTGGAGTGGGTCGCAACCATTAGTGGTGGTGGTCGTTACACCTACTATCCAGACAGTGTG AAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCGGIC TGAGGTCTGAGGACACAGCCATGTATTACTGTGCAAGTAATTACTACGGTTTTGACTACTGGGG CCAAGGCACCACTCTCACAGTCTCTTCA (SEQ ID NO: 15) MEIC729LC.3 Variable light chain region amino acid sequence: DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTG SGSGTDFTLTISNVQSEDLADYFCQQYSSYPWTFGGGTKLEIK (SEQ ID NO: 16) MEIC729LC.3 Variable light chain region nucleic acid sequence: GATATIGTGATGACCCAGICTCACAAATTCATGTCCACATCAGTAGGAGACAGGGICAGCATCA CCTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGCCIGGTATCAACAGAAACCAGGGCAATC TCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTICACGGGC AGTGGATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGGCAGATT ATTTCTGTCAGCAATATAGCAGCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAA AC

m136-M14-MHC724 mIgG2a/K

(SEQ ID NO: 17) MEIC724HC.3 Variable heavy chain region amino acid sequence: KVMLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWVATISGGGRDIYYADTV KGRFTISRDNAKNTLYLQMSSLRSEDTALYFCARLYLGFDYWGQGTTLTVSS (SEQ ID NO: 18) MEIC724HC.3 Variable heavy chain region nucleic acid sequence: AAAGTGATGCTGGTGGAGICTGGGGGAGACTTAGTGAAGCCTGGAGGGICCCTGAAACTCTCCT GTGCAGCCTCTGGATTCACTTTCAGTAGCTATGGCATGTCTTGGGTTCGCCAGACTCCGGAGAA GAGGCTGGAGTGGGTCGCAACCATTAGTGGTGGTGGTAGAGACATCTACTACGCAGACACTGTG AAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTACAAATGAGCAGTC TGAGGTCTGAGGACACGGCCTTGTATTTCTGTGCAAGGCTCTACCTGGGGTTTGACTACTGGGG CCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 19) MEIC724LC.1 Variable light chain region amino acid sequence: DIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLIYAATSLADGVPSRFSG SGSGTKFSFKISSLQAEDFVSYYCQQLYSIPWTFGGGTKLEIK (SEQ ID NO: 20) MEIC724LC.1 Variable light chain region nucleic acid sequence: GACATICAGATGACCCAGICTCCTGCCTCCCAGTCTGCATCTCTGGGAGAAAGTGTCACCATCA CATGCCTGGCAAGTCAGACCATTGGTACATGGITAGCATGGTATCAGCAGAAACCAGGGAAATC TCCTCAGCTCCTGATTTATGCTGCAACCAGCTTGGCAGATGGGGTCCCATCAAGGTTCAGTGGT AGTGGATCTGGCACAAAATTITCTTICAAGATCAGCAGCCTACAGGCTGAAGATTITGTAAGTT ATTACTGTCAACAACTITACAGTATTCCGTGGACATTCGGIGGAGGCACCAAGCTGGAAATCAA AC

Binding of mouse antibodies m136-M13, m136-M19, m245-M3, m245-M5 and m136-M14 to human PD-1 was confirmed by ELISA (FIG. 1). Briefly, human PD-1-Fc (R and D systems, Minneapolis, Minn.) was adsorbed to the wells of a 96-well ELISA plate. Purified anti-PD-1 antibodies were applied to the plate in serial dilution and allowed to bind. Plates were washed with PBST (PBS, pH 7.2+0.05% Tween-20). Bound antibody was detected with an anti-mouse IgG-HRP conjugate (Sigma, St Louis, Mo.) and visualized with the chromogenic substrate TMB (Thermo Scientific, Rockford, Ill.). Plots were generated in Prizm (Sigma Plot) and the data were fit to a model of single site saturation binding. Anti-PD-1 antibodies nivolumab (NV1) and/or pembrolizumab (PM1) were used as positive controls in binding assays with anti-human IgG-HRP conjugate, FAb-specific (Sigma, St Louis, Mo.) for detection. The K_(d) for each antibody tested is shown in Table 10 below:

TABLE 10 K_(d) values for tested antibodies: Clone K_(d), nM 245-M3  0.22 245-M5  0.34 136-M13 0.19 136-M19 0.23 136-M14 0.19 NV1 nivo 0.28 PM1 pembro 0.35

Binding of mouse antibodies m136-M13, m136-M19, m245-M3, m245-M5 and m136-M14 to human PD-1 inhibited PD-1 binding to PDL1 and PDL2 in an inhibition ELISA assay (FIGS. 2-3). Inhibition ELISAs were performed as follows. Human PD-1-Fc (R and D systems, Minneapolis, Minn.) was adsorbed to the wells of a 96-well ELISA plate. Purified anti-PD-1 antibodies were applied to the plate in serial dilution in the presence of 2 nM biotinylated PD-L1 or 2 nM biotinylated PDL2. Binding of biotinylated PD-L1 and PD-L2 was detected by Pierce™ Streptavidin-poly HRP conjugate (Thermo Scientific, Rockford, Ill.) and visualized with TMB. Plots were generated in Prizm (Sigma Plot) and the data were fit to a model of single site competition binding and an IC₅₀ was determined. The IC₅₀ values for the M13 and M14 antibodies are shown in Table 11 below:

TABLE 11 IC₅₀ values for antibodies tested IC50 (nM) hPDL1/Fc hPDL2/Fc M13 3.3 2   M14 4.8 2.8

Example 2. Production and Testing of Humanized Anti-PD-1 Antibodies

This example demonstrates that mouse antibodies of the disclosure that bind human PD-1 can be converted to humanized IgG antibodies that retain PD-1 binding and inhibition of PDL1 and PDL2 binding to PD-1.

Variable domains of the mouse anti-PD-1 antibodies produced as described in Example 1 were humanized and expressed as full length hIgG4/hKappa antibodies. Fully human IgGs anti-PD-1 antibodies were expressed from transiently transfected HEK-293 cells and purified from the culture supernatant by Protein A chromatography.

The sequences of the humanized antibodies used in the studies presented herein are shown below:

(SEQ ID NO: 21) PD-1 A Hv Variable heavy chain region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVAYISNSGGNAHYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREDYGTSPFVYWGQGTLVTVSS (SEQ ID NO: 22) PD-1 A Hv Variable heavy chain region nucleic acid sequence: GAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCT GTGCCGCCAGCGGCTTCACCTTTAGCGGCTACGCCATGAGCTGGGTGCGCCAGGCTCCTGGCAA AGGCCTGGAATGGGTGGCCTACATCAGCAACAGCGGCGGCAATGCCCACTACGCCGATAGCGTG AAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCC TGCGGGCCGAGGACACCGCCGTGTACTACTGCACCAGAGAGGACTACGGCACCAGCCCCTTCGT GTATTGGGGCCAGGGTACCCTCGTGACCGTCTCCTCA (SEQ ID NO: 23) PD-1 Ab Hv Variable heavy chain region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSYISNSGGNAHYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEDYGTSPFVYWGQGTLVTVSS (SEQ ID NO: 24) PD-1 Ab Hv Variable heavy chain region nucleic acid sequence: GAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCT GTGCCGCCAGCGGCTTCACCTTTAGCGGCTACGCCATGAGCTGGGTGCGCCAGGCTCCTGGCAA AGGCCTGGAATGGGTGAGTTACATCAGCAACAGCGGCGGCAATGCCCACTACGCCGATAGCGTG AAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCC TGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGAGGACTACGGCACCAGCCCCTTCGT GTATTGGGGCCAGGGTACCCTCGTGACCGTCTCCTCA (SEQ ID NO: 25) PD-1 Ae Hv Variable heavy chain region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVAYISNSGGNTHYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDYGTSPFVYWGQGTLVTVSS (SEQ ID NO: 26) PD-1 Ae Hv Variable heavy chain region nucleic acid sequence: GAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCT GTGCCGCCAGCGGCTTCACCTTTAGCGGCTACGCCATGAGCTGGGTGCGCCAGGCTCCTGGCAA AGGCCTGGAATGGGTGGCCTACATCAGCAACAGCGGCGGCAATACCCACTACGCCGATAGCGTG AAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCC TGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGAGGACTACGGCACCAGCCCCTTCGT GTATTGGGGCCAGGGTACCCTCGTGACCGTCTCCTCA (SEQ ID NO: 27) PD-1 Af Hv Variable heavy chain region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVAYISNSGGNTHYADSL KGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCAREDYGTSPFVYWGQGTLVTVSS (SEQ ID NO: 28) PD-1 Af Hv Variable heavy chain region nucleic acid sequence: GAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCT GTGCCGCCAGCGGCTTCACCTTTAGCGGCTACGCCATGAGCTGGGTGCGCCAGGCTCCTGGCAA AGGCCTGGAATGGGTGGCCTACATCAGCAACAGCGGCGGCAATACCCACTACGCCGATAGCCTG AAGGGCCGGTTCACCGTCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCC TGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGAGGACTACGGCACCAGCCCCTTCGT GTATTGGGGCCAGGGTACCCTCGTGACCGTCTCCTCA (SEQ ID NO: 29) PD-1 Ba Hv Variable heavy chain region amino acid sequence: QVQLVQSGAEVKKPGASVKMSCKASGYTFTDYYMDWVRQAPGQGLEWIGYIYPKNGGSSYAQKF QGRATLTVDTSTSTAYMELSSLRSEDTAVYYCARKVVATDYWGQGTLLTVSS (SEQ ID NO: 30) PD-1 Ba Hv Variable heavy chain region nucleic acid sequence: CAGGIGCAGCTGGTGCAGICIGGCGCCGAAGTGAAGAAACCAGGCGCCAGCGTGAAGATGAGCT GCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGGACTGGGTGCGCCAGGCCCCTGGACA GGGACTGGAATGGATCGGCTACATCTACCCCAAGAACGGCGGCAGCAGCTACGCCCAGAAGTTC CAGGGCAGAGCCACCCTGACCGTGGACACCAGCACAAGCACCGCCTACATGGAACTGAGCAGCC TGCGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGAAAGGIGGTGGCCACAGACTACTGGGG CCAGGGTACCCTGCTGACCGTGTCTAGT (SEQ ID NO: 31) PD-1 Bb Hv Variable heavy chain region amino acid sequence: QVQLVQSGAEVKKPGASVKMSCKASGYTFTDYYMDWVRQAPGQGLEWIGYIYPKNGGSSYAQKF QGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARKVVATDYWGQGTLLTVSS (SEQ ID NO: 32) PD-1 Bb Hv Variable heavy chain region nucleic acid sequence: CAGGIGCAGCTGGTGCAGICIGGCGCCGAAGTGAAGAAACCAGGCGCCAGCGTGAAGATGAGCT GCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGGACTGGGTGCGCCAGGCCCCTGGACA GGGACTGGAATGGATCGGCTACATCTACCCCAAGAACGGCGGCAGCAGCTACGCCCAGAAGTTC CAGGGCAGAGCCACCCTGACCGTGGACAAGAGCACCAGCACCGCCTACATGGAACTGAGCAGCC TGCGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGAAAGGIGGTGGCCACAGACTACTGGGG CCAGGGTACCCTGCTGACCGTGTCTAGT (SEQ ID NO: 33) PD-1 C Hy Variable heavy chain region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVAYISNGGGDTHYADSL KGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCARENYGTSPFVYWGQGTLVTVSS (SEQ ID NO: 34) PD-1 C Hy Variable heavy chain region nucleic acid sequence: GAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCT GTGCCGCCAGCGGCTICACCTICAGCAACTACGCCATGAGCTGGGTGCGCCAGGCCCCTGGAAA AGGCCTGGAATGGGTGGCCTACATCAGCAACGGCGGAGGCGATACCCACTACGCCGATAGCCTG AAGGGCCGGTTCACCGTGTCCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCC TGCGGGCCGAGGACACCGCCGTGTACTATTGCGCCAGAGAGAACTACGGCACCAGCCCCTTCGT GTACTGGGGCCAGGGTACCCTCGTGACCGTGTCCTCT (SEQ ID NO: 35) PD-1 Ca Hy Variable heavy chain region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVAYISNQGGDTHYADSL KGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCARENYGTSPFVYWGQGTLVTVSS (SEQ ID NO: 36) PD-1 Ca Hy Variable heavy chain region nucleic acid sequence: GAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCT GTGCCGCCAGCGGCTICACCTICAGCAACTACGCCATGAGCTGGGTGCGCCAGGCCCCTGGAAA AGGCCTGGAATGGGTGGCCTACATCAGCCAAGGCGGAGGCGATACCCACTACGCCGATAGCCTG AAGGGCCGGTTCACCGTGTCCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCC TGCGGGCCGAGGACACCGCCGTGTACTATTGCGCCAGAGAGAACTACGGCACCAGCCCCTTCGT GTACTGGGGCCAGGGTACCCTCGTGACCGTGTCCTCT (SQ ID NO: 37) PD-1 D Hv Variable heavy chain region amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAHSGFSFSSYDMSWVRQAPGKGLEWVATISGGGRYTYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASNYYGFDYWGQGTLLTVSS (SEQ ID NO: 38) PD-1 D Hv Variable heavy chain region nucleic acid sequence: GAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCT GTGCCCACAGCGGCTTCAGCTTCAGCAGCTACGACATGAGCTGGGTGCGCCAGGCCCCTGGCAA AGGACTGGAATGGGTGGCCACAATCAGCGGCGGAGGCCGGTACACCTACTACGCCGATAGCGTG AAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCC TGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGCAACTACTACGGCTTCGACTACTGGGG CCAGGGTACCCTGCTGACCGTGTCATCT (SEQ ID NO: 39) PD-1 1.0 Lv Variable light chain region amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPS RFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK (SEQ ID NO: 40) PD-1 1.0 Lv Variable light chain region nucleic acid sequence: GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCA CCTGTAGAGCCAGCGAGAGCGTGGACAACTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAA GCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGC AGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAA GCTGGAAATCAAG (SEQ ID NO: 41) PD-1 1.1 Lv Variable light chain region amino acid sequence: DIQLTQSPSSLSVSVGDRATITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPS RFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK (SEQ ID NO: 42) PD-1 1.1 Lv Variable light chain region nucleic acid sequence: GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCCGTGTCCGTGGGCGACAGAGCCACCATCA CCTGTAGAGCCAGCGAGAGCGTGGACAACTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAA GCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGC AGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAA GCTGGAAATCAAG (SEQ ID NO: 43) PD-1 1.2 Lv Variable light chain region amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASESVDQYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPS RFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWFGQGTKLEIK (SEQ ID NO: 44) PD-1 1.2 Lv Variable light chain region nucleic acid sequence: GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCA CCTGTAGAGCCAGCGAGAGCGTGGACCAATACGGCATCAGCTTCATGAACTGGTTCCAGCAGAA GCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGC AGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAA GCTGGAAATCAAG (SEQ ID NO: 45) PD-1 1.4 Lv Variable light chain region amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASESVDSYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPS RFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK (SEQ ID NO: 46) PD-1 1.4 Lv Variable light chain region nucleic acid sequence: GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCA CCTGTAGAGCCAGCGAGAGCGTGGACAGTTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAA GCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGC AGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAA GCTGGAAATCAAG (SEQ ID NO: 47) PD-1 1.5 Lv Variable light chain region amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPS RFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK (SEQ ID NO: 48) PD-1 1.5 Lv Variable light chain region nucleic acid sequence: GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCA CCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAA GCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGC AGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAA GCTGGAAATCAAG (SEQ ID NO: 49) PD-1 1.6 Lv Variable light chain region amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASDQGSGVPS RFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK (SEQ ID NO: 50) PD-1 1.6 Lv Variable light chain region nucleic acid sequence: GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCA CCTGTAGAGCCAGCGAGAGCGTGGACAACTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAA GCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCGATCAGGGCAGCGGCGTGCCAAGC AGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAA GCTGGAAATCAAG (SEQ ID NO: 51) PD-1 1.7 Lv Variable light chain region amino acid sequence: DIQLTQSPSSLSVSVGDRATITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPS RFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK (SEQ ID NO: 52) PD-1 1.7 Lv Variable light chain region nucleic acid sequence: GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCCGTGTCCGTGGGCGACAGAGCCACCATCA CCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAA GCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGC AGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAA GCTGGAAATCAAG (SEQ ID NO: 53) PD-1 1.9 Lv Variable light chain region amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPS RFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKVEIK (SEQ ID NO: 54) PD-1 1.9 Lv Variable light chain region nucleic acid sequence: GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCA CCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAA GCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGC AGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAA GGTGGAAATCAAG (SEQ ID NO: 55) PD-1 1.10 Lv Variable light chain region amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPS RFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPYTFGQGTKLEIK (SEQ ID NO: 56) PD-1 1.10 Lv Variable light chain region nucleic acid sequence: GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCA CCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAA GCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGC AGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTACACCTTTGGCCAGGGTACCAA GCTGGAAATCAAG (SEQ ID NO: 57) PD-1 2 Lv Variable light chain region amino acid sequence: DIQMTQSPSSLSASVGDRVTMTCKSSQSLLYSSNQKNYLAWYQQKPGKAPKLLIFWASIRESGV PSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSDSYPWTFGQGTKLEIK (SEQ ID NO: 58) PD-1 2 Lv Variable light chain region nucleic acid sequence: GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTGGGCGATAGAGTGACCATGA CCTGCAAGAGCAGCCAGAGCCTGCTGTACTCCAGCAACCAGAAGAACTACCTGGCCTGGTATCA GCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTTCTGGGCCAGCATCCGGGAAAGCGGCGTG CCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGC CCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCGACAGCTACCCCTGGACCTTTGGCCAGGG TACCAAGCTGGAAATCAAG (SEQ ID NO: 59) PD-1 4 Lv Variable light chain region amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSG SGSGTDFTLTISSVQPEDFATYYCQQYSSYPWTFGQGTKLEIK (SEQ ID NO: 60) PD-1 4 Lv Variable light chain region nucleic acid sequence: GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCA CATGCAAGGCCAGCCAGGACGTGGGAACAGCCGTGGCCTGGTATCAGCAGAAGCCTGGCAAGGC CCCCAAGCTGCTGATCTACTGGGCCAGCACCAGACACACCGGCGTGCCCAGCAGATTTTCTGGC AGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGCCCGAGGACTTCGCCACCT ACTACTGCCAGCAGTACAGCAGCTACCCCTGGACCTTTGGCCAGGGTACCAAGCTGGAAATCAA G (SEQ ID NO: 61) Kappa constant region amino acid sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 62) Kappa constant region nucleic acid sequence: CGTACGGIGGCTGCACCATCTGICTICATCTICCCGCCATCTGATGAGCAGTTGAAATCTGGAA CTGCCTCTGTTGTGTGCCTGCTGAATAACTICTATCCCAGAGAGGCCAAAGTACAGTGGAAGGT GGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC ACCTACAGCCICAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGICTACG CCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTICAACAGGGGAGAGTG T (SEQ ID NO: 63) hIgG4 S228P amino acid sequence: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 64) hIgG4 S228P nucleic acid sequence: GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTGTAGCAGAAGCACCAGCGAGTCTA CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTC AGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCAAGACCTACACCTGTAACGTGG ACCACAAGCCCAGCAACACCAAGGTGGACAAGCGGGTGGAATCTAAGTACGGCCCTCCCTGCCC TCCTTGCCCAGCCCCTGAATTICTGGGGGGACCGTCAGICTICCTCTICCCCCCAAAACCCAAG GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGAAG ACCCTGAGGICCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC GCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC TGGCTGAATGGCAAGGAGTACAAGTGCAAGGICTCCAACAAAGGCCTGCCCAGCTCCATCGAGA AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGIGTACACCCTGCCCCCATCCCA GGAGGAGATGACCAAGAACCAGGICAGCCTGACCTGCCTGGICAAAGGCTICTATCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGC TGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGACTCACCGTGGACAAGAGCAGGTGGCAGGA AGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC CTCTCCCTGTCTCTGGGTAAA

These variable heavy chain regions (VH) and variable light chain regions (VL) can be used in a variety of combinations to produce anti-PD-1 antibodies of the disclosure. For example, the antibody referred to herein as A1.0 includes the VH sequence of SEQ ID NO: 21 and the VL sequence of SEQ ID NO: 39, the A1.5 antibody includes the VH sequence of SEQ ID NO: 21 and the VL sequence of SEQ ID NO: 47; the antibody referred to herein as C1.1 includes the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 41, and so on.

As shown in FIGS. 4-5, humanized anti-PD-1 antibodies bound to hPD-1 in a standard ELISA in a manner similar to that of nivolumab and/or pembrolizumab, and binding of the humanized antibodies inhibited the binding of PD-1 to PDL1 and PDL2 (FIGS. 6-9) in inhibition ELISAs. ELISAs were performed as follows. For hPD-1 binding ELISAs, human PD-1-Fc (R and D systems, Minneapolis, Minn.) was adsorbed to the wells of a 96-well ELISA plate. Purified anti-PD-1 antibodies were applied to the plate in serial dilution and allowed to bind. Bound antibody was detected with an anti-human IgG-HRP conjugate (FAb-specific) (Sigma, St Louis, Mo.) and visualized with the chromogenic substrate TMB (Thermo Scientific, Rockford, Ill.). Plots were generated in Prizm (Sigma Plot) and the data were fit to a model of single site saturation binding. For ligand inhibition ELISAs, human PD-1-Fc (R and D systems, Minneapolis, Minn.) was adsorbed to the wells of a 96-well ELISA plate. Purified anti-PD-1 antibodies were applied to the plate in serial dilution in the presence of 2 nM biotinylated hPD-L1 or 2 nM biotinylated hPD-L2. Pierce™ Streptavidin-poly HRP conjugate (Thermo Scientific, Rockford, Ill.) and visualized with TMB. Plots were generated in Prizm (Sigma Plot) and the data were fit to a model of single site competition binding and an IC₅₀ was determined.

Example 3: Anti-PD-1 Antibodies Shows Specificity in Binding

Example 3 shows that humanized anti-PD-1 antibody groups A1 and C1 of the disclosure bind specifically to hPD-1 by plate ELISA.

The binding of anti-PD-1 antibody A1.5 of the disclosure was highly specific to hPD-1-Fc in a standard ELISA against a panel of numerous human and mouse proteins (FIG. 10). Binding of anti-PD-1 A1.5 was detected with an anti-human IgG-HRP conjugate (FAb-specific) (Sigma, St Louis, Mo.) and visualized with the chromogenic substrate TMB (Thermo Scientific, Rockford, Ill.). Plots were generated in Prizm (Sigma Plot) and the data were fit to a model of single site saturation binding.

Example 4: Anti-PD-1 Epitope Binding

This example compares the epitopes bound by humanized anti-PD-1 antibodies of the disclosure, nivolumab, and pembrolizumab.

Nivolumab and pembrolizumab anti-PD-1 antibodies each bind human PD-1 and such binding by each inhibits the binding of PD-1 to PDL1 and PDL2. To map the epitopes of humanized groups A1 and C1 antibodies of the disclosure, anti-PD-1 inhibition ELISAs were performed, and compared with nivolumab and pembrolizumab inhibition. Dilution series of nivolumab, pembrolizumab, A1 and C1 antibodies were incubated in the presence of biotinylated nivolumab FAb or biotinylated pembrolizumab FAb at a concentration of 0.3 nM in a standard plate ELISA format with hPD-1-Fc (R & D systems, Minneapolis, Minn.). Binding of biotinylated FAb was detected by Pierce™ Streptavidin-poly HRP conjugate (Thermo Scientific, Rockford, Ill.) and visualized with TMB (Thermo Scientific, Rockford Ill.) and 1N HCl. Antibodies belonging to the A1 and C1 groups of the disclosure blocked biotinylated Pembrolizumab FAb binding to PD-1 similarly to Pembrolizumab antibody and more completely than nivolumab antibody (FIG. 11A). The same A1 and C1 antibodies of the disclosure blocked biotinylated nivolumab binding similarly to nivolumab antibody and more completely than pembrolizumab antibody (FIG. 11B). The data in FIGS. 11A and 11B show that A1 and C1 antibodies of the disclosure completely block both nivolumab and pembrolizumab, whereas nivolumab and pembrolizumab incompletely block each other.

Example 5: Anti-PD-1 A1.5 Enhances CMV-Stimulated Cytokine Secretion by PBMCs from a CMV-Positive Donor

In this example, peripheral blood mononuclear cells from a CMV-positive donor were incubated in the presence of CMV viral lysate and anti-PD-1 antibodies of the disclosure to assess the effect of such anti-PD-1 antibodies on interferon gamma (IFN-gamma, IFNg, IFN-g, IFNγ, or IFN-γ) cytokine secretion.

PBMCs from a CMV-positive donor (Hemacare) were plated at 2×10⁵ cells/well in the presence of CMV viral lysate (Astarte) and either anti-PD-1 antibody A1.5 of the disclosure or a hIgG4 isotype control antibody. After four days, supernatant was removed from each well, and IFN-gamma levels were assayed using IFN-gamma ELISA kit (Life Technologies, Carlsbad, Calif.) (FIG. 12). Anti-PD-1 antibody A1.5 increased CMV-stimulated IFN-gamma secretion compared with control hIgG4 and with potency similar to anti-PD-1 nivolumab and pembrolizumab antibodies.

Example 6: Anti-PD-1 Antibodies Bind to Monomeric hPD-1 with High Affinity and Slow Dissociation Kinetics

In this example, humanized anti-PD-1 antibodies of the disclosure are shown to bind with high affinity and slow dissociation to monomeric PD-1.

Activatable antibodies can be activated singly, creating a monovalent binding moiety, or doubly, creating a bivalent binding moiety. Single-arm activation of antibodies with differing avidities can favor binding and biological activity of activatable antibodies with higher monovalent affinities. Anti-PD-1 antibodies A1.5 and Bba2 of the disclosure, as well as nivolumab and pembrolizumab were immobilized at equivalent densities on Forte-Bio Octet BioLayer Inferometry (Pall ForteBio, Menlo Park, Calif.) sensors and allowed to bind to a serial dilution of human PD-1-His (R & D systems, Minneapolis, Minn.) in solution. Kinetic analysis was performed with ForteBio Data Analysis software. Results (FIG. 13) show PD-1 antibodies of the disclosure bind monomeric PD-1 with similar or higher affinities and similar or slower dissociation constants than nivolumab or pembrolizumab.

Example 7: Activatable Anti-PD-1 M13/A1.4/A1.5 Antibody Masking Moieties

This example describes identification of masking moieties (MM) that reduce binding of anti-PD-1 antibodies of the disclosure to their target.

Anti-PD-1 antibodies m136-M13, A1.4 and A1.5 were used to screen libraries using a method similar to that described in PCT International Publication Number WO 2010/081173, published 15 Jul. 2010. The screening consisted of one round of MACS and five rounds of FACS sorting. For the initial MACS, approximately 2×10¹¹ cells were incubated with m136-M13 antibody at a concentration of 100 nM, and 6×10⁶ binders were collected using Protein-G Dynabeads (Invitrogen). FACS rounds were conducted labeling cells with DyLight 650 (Thermo-Fisher) labeled m136-M19 antibody for FACS rounds 1-4 as follows: 100 nM FACS round 1 (F1), 10 nM FACS round 2 (F2), 2 nM FACS round 3 (F3) and 1 nM FACS round 4 (F4), with increasingly small percentages of binders as assessed by fluorescence collected at each round. For FACS round 5 (F5), cells were labeled with 1 nM DyLight 650 labeled A1.5 antibody and the brightest 0.2-4% of cells were collected. Individual peptide clones from F3, F4 and F5 were identified by sequence analysis and subsequently verified for their ability to bind DyLight-650 A1.4 or DyLight-650 A1.5.

The sequences of the anti-PD-1 m136-M13, A1.4 and A1.5 masking moieties are listed in Table 12 (masking moiety PD001 is also referred to in the disclosure as PD01 and/or PD-01; masking moiety PD002 is also referred to herein as PD02 and/or PD-02, and so on):

TABLE 12 Masking Moieties Mask Amino Acid Sequence SEQ ID NO: PD001 AMSGCSWSAFCPYLA 66 PD002 DVNCAIWYSVCTTVP 67 PD003 LVCPLYALSSGVCMG 68 PD004 SVNCRIWSAVCAGYE 69 PD005 MLVCSLQPTAMCERV 70 PD006 APRCYMFASYCKSQY 71 PD007 VGPCELTPKPVCNTY 72 PD008 ETCNQYERSSGLCFA 73 PD009 APRTCYTYQCSSFYT 74 PD010 GLCSWYLSSSGLCVD 75 PD011 VPWCQLTPRVMCMWA 76 PD012 NWLDCQFYSECSVYG 77 PD013 SCPLYVMSSFGGCWD 78 PD014 MSHCWMFSSSCDGVK 79 PD015 VSYCTWLIEVTCLRG 80 PD016 VLCAAYALSSGICGG 81 PD017 TTCNLYQQSSMFCNA 82 PD018 APRCYMFASYCKSQY 83 PD019 PCDQNPYFYPYVCHA 84 PD020 SVCPMYALSSMLCGA 85 PD021 LSVECYVFSRCSSLP 86 PD022 FYCTYLVSLTCHPQ 87 PD023 SMAGCQWSSFCVQRD 88 PD024 IYSCYMFASRCTSDK 89 PD025 SRCSVYEVSSGLCDW 90 PD026 GMCSAYAYSSKLCTI 91 PD027 MTTNTCNLLCQQFLT 92 PD028 FQPCLMFASSCFTSK 93 PD029 WNCHPAGVGPVFCEV 94 PD030 ALCSMYLASSGLCNK 95 PD031 NYLSCQFFQNCYETY 96 PD032 GWCLFSDMWLGLCSA 97 PD033 EFCARDWLPYQCSSF 98 PD034 TSYCSIEHYPCNTHH 99 PD035 PYICSSFPLDCQAGQ 100 PD036 VGCEWYMSSSGMCSR 101 PD037 EVCGGCSMQSVSCWP 102 PD038 FTECQLSPKAICMSN 103 PD039 KYCLFSEYVEGTCLN 104 PD040 SGCPMYAWGWDECWR 105 PD041 VDCPWYASSSAICSR 106 PD042 DMLLCQIRGSCAAWG 107 PD043 ECHPYQASASLWCGY 108 PD044 MMMGCMWSAWCPPSR 109 PD045 NAYFRCSLMCNMIMF 110 PD046 ACCKESVHSVHDCKR 111 PD047 ACIGINSYMSNYCYL 112 PD048 ANCSFLELTNKFCTI 113 PD049 AYCSYLMFASNPCII 114 PD050 CFTSKCPCLCYSLLA 115 PD051 CLCRDINCWLGCSKT 116 PD052 CWCDIYCSPYQCSSF 117 PD053 DCIYYYQQSANLCSY 118 PD054 DCTGVNYYIDKHCTN 119 PD055 DECHGYLRSSGLCGG 120 PD056 DICSAYAASSGFCYY 121 PD057 DIICVLTPTAWCGRT 122 PD058 DNCCMYCSWWIACRD 123 PD059 DSCQWYMLSADLCGT 124 PD060 DSVCFSSSSFLCHKS 125 PD061 DTMCAIWWTVCSGGR 126 PD062 ECTYQTSSFHEACMS 127 PD063 EGCNLYERSSYGCNN 128 PD064 EGCTAFAMSAGICGG 129 PD065 EQSCSLTPIAFCWSE 130 PD066 EWCNAYISSSKLCST 131 PD067 FEVCYMFASACRNGM 132 PD068 FSCSWYAESSSLCDI 133 PD069 FVCQMFEASSGLCGG 134 PD070 FYCPCCMFASSCGSR 135 PD071 FYCSYLPGASHQCSH 136 PD072 FYCSYLYMCEVCCYE 137 PD073 GFCTQHTVLTWCPTS 138 PD074 GSCPSYAVSAGLCYA 139 PD075 GSQCFLTPTAFCTHT 140 PD076 GTCHPYMQSSKICNN 141 PD077 GVECFVFTGGCGGYG 142 PD078 HELCNGHWVPCCWAY 143 PD079 ICDSYYAVSSGLCLL 144 PD080 IGCAWYVSSAGWCSP 145 PD081 INLCWMFASECGEHH 146 PD082 KCWLAEMTNLEHCNM 147 PD083 KHCSDFAYSSRLCDR 148 PD084 KVCSSYASSSGLCGW 149 PD085 LDSCYMFASYCVQAV 150 PD086 LLACHPIFVTVCQTR 151 PD087 LLSCPYNPEHVCHTS 152 PD088 LMCSLYALSSNLCGR 153 PD089 LMWCVLFLWSWCCRI 154 PD090 LPICHLTPTAVCTHI 155 PD091 LSNMCLAFGSCLYAW 156 PD092 LSRCHPIWYTICQNP 157 PD093 LTQCMSVHKECGGYE 158 PD094 LVNCRIWSWVCEEAT 159 PD095 LYCSWYQMSSAVCKE 160 PD096 MECGWYALSARFCEV 161 PD097 MTCSPYAMSAHFCNE 162 PD098 MVCSLYAYSASLCGA 163 PD099 NALCWSTFSWWCDMD 164 PD-100 NFTCMLTPKAYCVQT 165 PD-101 NGACIFTLSWCTNKT 166 PD-102 NGCELYAAASGLCRT 167 PD-103 NIECSVFGRCCCDNY 168 PD-104 PACRPMFWNRSCDNI 169 PD-105 PCRVSNMFFPYNCLD 170 PD-106 PIMCMLLPESYCWIW 171 PD-107 PQSCYMFASLCMPNG 172 PD-108 PRCPQGLPLYQCSSF 173 PD-109 PSVECLVFKRCYALP 174 PD-110 PVCQRSATIYNCNWF 175 PD-111 QCAAYYISSFGGCSN 176 PD-112 QFGCFMLARDFCGTY 177 PD-113 QMMCPYNPEHKCHQK 178 PD-114 QRECWMFASSCNSKN 179 PD-115 QSNMCTTYICSSFNY 180 PD-116 QSRCHSLAPYLCSSF 181 PD-117 RAYCSLLFADSCNNN 182 PD-118 RCIGINQYIDSNCYN 183 PD-119 RLSCFMFASQCALEF 184 PD-120 RQCIILMNHRQCFFK 185 PD-121 RSCTPYMMSSSLCNT 186 PD-122 RYCHYWKMPYECSSF 187 PD-123 SCVSLSWFDMLKCYE 188 PD-124 SDNCEIWWTVCSAAM 189 PD-125 SFCWSYLVSSGLCGV 190 PD-126 SMCMNNYGTTIMCGN 191 PD-127 SMVGCGWSTFCPSRG 192 PD-128 SSLHCANGHTCPFCL 193 PD-129 SVCSYYEESSGICSP 194 PD-130 SWCGWYAASSGVCAL 195 PD-131 TCISQTIDSYLNCVN 196 PD-132 TFCNLYTKSSNICMS 197 PD-133 TYCVFHEYLDNTCNN 198 PD-134 VATGCPNLMLCGSWP 199 PD-135 VEYCSLLLGNRCDYW 200 PD-136 VGCNMYLMSAGLCVD 201 PD-137 VLYCSWDSGTCVGSH 202 PD-138 VMFSCYYLETCAPGV 203 PD-139 VRIGLCPESCLVSGF 204 PD-140 VTCTYYATSSSLCNT 205 PD-141 VTGCILLPKAWCWGD 206 PD-142 VWCSIYEYSSNLCSR 207 PD-143 WMLECQYNNTCNNMT 208 PD-144 WPCSPLEYYNNICNV 209 PD-145 WTYDCHLNQTCPTYY 210 PD-146 YCSINMYLIGGNCMY 211 PD-147 YFCSLYANSAGFCGG 212 PD-148 YVSCYMFSSSCPSTW 213

Example 8: Activatable Anti-PD-1 A1.4 and A1.5 Antibodies

This example describes examples of activatable anti-PD-1 A1.4 and A1.5 antibodies of the disclosure.

Activatable anti-PD-1 A1.4 antibodies comprising an anti-PD-1 M13 masking moiety, a cleavable moiety, and an anti-PD-1 A1.4 antibody of the disclosure and activatable anti-PD-1 A1.5 antibodies comprising an anti-PD-1 M13 or an anti-PD-1 A1.5 masking moiety, a cleavable moiety selected from the group consisting of a cleavable moiety, and an anti-PD-1 A1.5 antibody were produced according to methods similar to those described in PCT Publication Nos. WO 2009/025846 and WO 2010/081173, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the cleavable moiety was selected from the group consisting of a cleavable moiety referred to herein as “2001” and comprising the sequence ISSGLLSGRSDNH (SEQ ID NO: 214) and a cleavable moiety referred to herein as “3001” and comprising the sequence AVGLLAPPGGLSGRSDNH (SEQ ID NO: 318). The amino acid and nucleic acid sequences of several activatable anti-PD-1 antibody variable domains of the disclosure are provided below. Antibodies were produced as hIgG4 containing a single amino acid substitution, S228P (Angal, et al. 1993. Mol Immunol 30:105-8.) HC and hK LC format.

In some embodiments, the activatable antibody also includes a spacer sequence. In some embodiments, the spacer is joined directly to the MM of the activatable antibody. In some embodiments, the spacer is joined directly to the MM of the activatable antibody in the structural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB. In some embodiments, the spacer joined directly to the N-terminus of MM of the activatable antibody is selected from the group consisting of QGQSGQG (SEQ ID NO: 362), QGQSGQ (SEQ ID NO: 913), QGQSG (SEQ ID NO: 914), QGQS (SEQ ID NO: 915), QGQ (SEQ ID NO: 916), QG (SEQ ID NO: 917), and Q. In some embodiments, the spacer includes at least the amino acid sequence QGQSGQG (SEQ ID NO: 362). In some embodiments, the spacer includes at least the amino acid sequence QGQSGQ (SEQ ID NO: 913). In some embodiments, the spacer includes at least the amino acid sequence QGQSG (SEQ ID NO: 914). In some embodiments, the spacer includes at least the amino acid sequence QGQS (SEQ ID NO: 915), In some embodiments, the spacer includes at least the amino acid sequence QGQ (SEQ ID NO: 916). In some embodiments, the spacer includes at least the amino acid sequence QG (SEQ ID NO: 917). In some embodiments, the spacer includes at least the amino acid residue Q. In some embodiments, the activatable antibody does not include a spacer sequence.

While the sequences shown below include the spacer sequence of SEQ ID NO: 362, those of ordinary skill in the art appreciate that the activatable anti-PD-1 antibodies of the disclosure can include any suitable spacer sequence, such as, for example, a spacer sequence selected from the group consisting of QGQSGQG (SEQ ID NO: 362), QGQSGQ (SEQ ID NO: 913), QGQSG (SEQ ID NO: 914), QGQS (SEQ ID NO: 915), QGQ (SEQ ID NO: 916), QG (SEQ ID NO: 917), and Q. Additional examples of spacers include GQSGQG (SEQ ID NO: 2042), QSGQG (SEQ ID NO: 2043), SGQG (SEQ ID NO: 2044), GQG(SEQ ID NO: 2045), QG(SEQ ID NO: 2046), and G. While the sequences shown below include the spacer sequence of SEQ ID NO: 362, those of ordinary skill in the art will also appreciate that activatable anti-PD-1 antibodies of the disclosure in some embodiments do not include a spacer sequence.

Activatable Anti-PD-1 Variable Domains:

[Spacer (SEQ ID NO: 362)]  [PD-1 1.4 PD001 2001 (SEQ ID NO: 919)] (SEQ ID NO: 215) [QGQSGQG][AMSGCSWSAFCPYLAGGGSSGGSISSGLLSGRSDNHG GGSDIQLTQSPSSLSASVGDRVTITCRASESVDSYGISFMNWFQQ KPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYY CQQSKDVPWTFGQGTKLEIK] PD-1 1.4 PD001 2001 Amino Acid Sequence: (SEQ ID NO: 1041) AMSGCSWSAFCPYLAGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDSYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)] [PD-1 1.4 PD001 2001 (SEQ ID NO: 920)] (SEQ ID NO: 216) [CAAGGCCAGTCTGGCCAAGGT][GCGATGAGTGGGTGCTCGTGGT CTGCTTTTTGCCCGTATTTGGCGGGAGGTGGCTCGAGCGGCGGCTC TATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGA TCCGACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGG GCGACAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACAGTTA CGGCATCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCC AAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCA GATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAG CATGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGAC GTGCCCTGGACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PD-1 1.4 PD002 2001 (SEQ ID NO: 921)] (SEQ ID NO: 217) [QGQSGQG][DVNCAIWYSVCTTVPGGGSSGGSISSGLLSGRSDNHG GGSDIQLTQSPSSLSASVGDRVTITCRASESVDSYGISFMNWFQQ KPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYY CQQSKDVPWTFGQGTKLEIK] PD-1 1.4 PD002 2001 Amino Acid Sequence: (SEQ ID NO: 1042) DVNCAIWYSVCTTVPGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDSYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.4 PD002 2001 (SEQ ID NO: 922)] (SEQ ID NO: 218) [CAAGGCCAGTCTGGCCAAGGT][GATGTTAATTGCGCTATTTGGTA TTCGGTGTGCACTACTGTTCCTGGAGGTGGCTCGAGCGGCGGCTC TATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGA TCCGACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGG GCGACAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACAGTTA CGGCATCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCC AAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCA GATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAG CATGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGAC GTGCCCTGGACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.4 PD003 2001 (SEQ ID NO: 923)] (SEQ ID NO: 219) [QGQSGQG][LVCPLYALSSGVCMGGGGSSGGSISSGLLSGRSDNHG GGSDIQLTQSPSSLSASVGDRVTITCRASESVDSYGISFMNWFQQ KPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYY CQQSKDVPWTFGQGTKLEIK] PD-1 1.4 PD003 2001 Amino Acid Sequence: (SEQ ID NO: 1043) LVCPLYALSSGVCMGGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDSYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.4 PD003 2001 (SEQ ID NO: 924)] (SEQ ID NO: 220) [PD-1 1.4 PD003 2001 (SEQ ID NO: 924)] [CAAGGCCAGTCTGGCCAAGGT][TTGGTTTGCCCTTTGTATGCATT GAGTTCTGGGGTGTGCATGGGGGGAGGTGGCTCGAGCGGCGGCTC TATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGA TCCGACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGG GCGACAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACAGTTA CGGCATCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCC AAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCA GATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAG CATGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGAC GTGCCCTGGACCT TTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.4 PD008 2001 (SEQ ID NO: 925)] (SEQ ID NO: 221) [QGQSGQG][ETCNQYERSSGLCFAGGGSSGGSISSGLLSGRSDNHG GGSDIQLTQSPSSLSASVGDRVTITCRASESVDSYGISFMNWFQQ KPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYY CQQSKDVPWTFGQGTKLEIK] PD-1 1.4 PD008 2001 Amino Acid Sequence: (SEQ ID NO: 1044) ETCNQYERSSGLCFAGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDSYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.4 PD008 2001 (SEQ ID NO: 926)] (SEQ ID NO: 222) [CAAGGCCAGTCTGGCCAAGGT][GAGACTTGCAATCAGTATGAGAG GTCGAGTGGTTTGTGCTTTGCGGGAGGTGGCTCGAGCGGCGGCTC TATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGA TCCGACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGG GCGACAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACAGTTA CGGCATCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCC AAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCA GATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAG CATGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGAC GTGCCCTGGACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.4 PD009 2001 (SEQ ID NO: 927)] (SEQ ID NO: 223) [QGQSGQG][APRTCYTYQCSSFYTGGGSSGGSISSGLLSGRSDNHG GGSDIQLTQSPSSLSASVGDRVTITCRASESVDSYGISFMNWFQQ KPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYY CQQSKDVPWTFGQGTKLEIK] PD-1 1.4 PD009 2001 Amino Acid Sequence: (SEQ ID NO: 1045) APRTCYTYQCSSFYTGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDSYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.4 PD009 2001 (SEQ ID NO: 928)] (SEQ ID NO: 224) [CAAGGCCAGTCTGGCCAAGGT][GCGCCGCGGACGTGCTATACGTA TCAGTGCTCTAGTTTTTATACTGGAGGTGGCTCGAGCGGCGGCTC TATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGA TCCGACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGG GCGACAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACAGTTA CGGCATCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCC AAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCA GATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAG CATGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGAC GTGCCCTGGACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.4 PD010 2001 (SEQ ID NO: 929)] (SEQ ID NO: 225) [QGQSGQG][GLCSWYLSSSGLCVDGGGSSGGSISSGLLSGRSDNHG GGSDIQLTQSPSSLSASVGDRVTITCRASESVDSYGISFMNWFQQ KPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYY CQQSKDVPWTFGQGTKLEIK] PD-1 1.4 PD010 2001 Amino Acid Sequence: (SEQ ID NO: 1046) GLCSWYLSSSGLCVDGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDSYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.4 PD010 2001 (SEQ ID NO: 930)] (SEQ ID NO: 226) [CAAGGCCAGTCTGGCCAAGGT][GGTCTTTGCAGTTGGTATCT TAGTAGTTCGGGTTTGTGCGTGGATGGAGGTGGCTCGAGCGGCGGCTCTA TCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCC GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGA CAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACAGTTACGGCA TCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTC CGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGC CCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGG ACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD01 2001 (SEQ ID NO: 931)] (SEQ ID NO: 227) [QGQSGQG][AMSGCSWSAFCPYLAGGGSSGGSISSGLLSGRSD NHGGGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKP GKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQ SKDVPWTFGQGTKLEIK] PD-1 1.5 PD01 2001 Amino Acid Sequence: (SEQ ID NO: 1047) AMSGCSWSAFCPYLAGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD001 2001 (SEQ ID NO: 932)] (SEQ ID NO: 228) [CAAGGCCAGTCTGGCCAAGGT][GCGATGAGTGGGTGCTCGTG GTCTGCTTTTTGCCCGTATTTGGCGGGAGGTGGCTCGAGCGGCGGCTCTA TCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCC GATATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGA CAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCA TCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTC CGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGC CCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGG ACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD002 2001 (SEQ ID NO: 933)] (SEQ ID NO: 229) [QGQSGQG][DVNCAIWYSVCTTVPGGGSSGGSISSGLLSGRSD NHGGGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKP GKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQ SKDVPWTFGQGTKLEIK] PD-1 1.5 PD002 2001 Amino Acid Sequence: (SEQ ID NO: 1048) DVNCAIWYSVCTTVPGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD002 2001 (SEQ ID NO: 934)] (SEQ ID NO: 230) [CAAGGCCAGTCTGGCCAAGGT][GATGTTAATTGCGCTATTTG GTATTCGGTGTGCACTACTGTTCCTGGAGGTGGCTCGAGCGGCGGCTCTA TCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCC GATATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGA CAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCA TCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTC CGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGC CCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGG ACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD003 2001 (SEQ ID NO: 935)] (SEQ ID NO: 231) [QGQSGQG][LVCPLYALSSGVCMGGGGSSGGSISSGLLSGRSD NHGGGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKP GKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQ SKDVPWTFGQGTKLEIK] PD-1 1.5 PD003 2001 Amino Acid Sequence: (SEQ ID NO: 1049) LVCPLYALSSGVCMGGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD003 2001 (SEQ ID NO: 936)] (SEQ ID NO: 232) [CAAGGCCAGTCTGGCCAAGGT][TTGGTTTGCCCTTTGTATGC ATTGAGTTCTGGGGTGTGCATGGGGGGAGGTGGCTCGAGCGGCGGCTCTA TCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCC GATATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGA CAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCA TCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTC CGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGC CCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGG ACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD004 2001 (SEQ ID NO: 937)] (SEQ ID NO: 233) [QGQSGQG][SVNCRIWSAVCAGYEGGGSSGGSISSGLLSGRSD NHGGGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKP GKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQ SKDVPWTFGQGTKLEIK] PD-1 1.5 PD004 2001 Amino Acid Sequence: (SEQ ID NO: 1050) SVNCRIWSAVCAGYEGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD004 2001 (SEQ ID NO: 938)] (SEQ ID NO: 234) [CAAGGCCAGTCTGGCCAAGGT][TCTGTGAATTGCCGGATTTG GTCGGCTGTTTGCGCGGGGTATGAGGGAGGTGGCTCGAGCGGCGGCTCTA TCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCC GATATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGA CAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCA TCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTC CGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGC CCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGG ACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD005 2001 (SEQ ID NO: 939)] (SEQ ID NO: 235) [QGQSGQG][MLVCSLQPTAMCERVGGGSSGGSISSGLLSGRSD NHGGGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKP GKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQ SKDVPWTFGQGTKLEIK] PD-1 1.5 PD005 2001 Amino Acid Sequence: (SEQ ID NO: 1051) MLVCSLQPTAMCERVGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD005 2001 (SEQ ID NO: 940)] (SEQ ID NO: 236) [CAAGGCCAGTCTGGCCAAGGT][ATGCTTGTGTGCTCGTTGCAGCCTAC TGCGATGTGCGAGCGGGTGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD006 2001 (SEQ ID NO: 941)] (SEQ ID NO: 237) [QGQSGQG][APRCYMFASYCKSQYGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD006 2001 Amino Acid Sequence: (SEQ ID NO: 941) APRCYMFASYCKSQYGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD006 2001 (SEQ ID NO: 942)] (SEQ ID NO: 238) [CAAGGCCAGTCTGGCCAAGGT][GCGCCTAGGTGCTATATGTTTGCGTC GTATTGCAAGAGTCAGTATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] PD-1 1.5 PD006 2001 Nucleic Acid Sequence: (SEQ ID NO: 942) GCGCCTAGGTGCTATATGTTTGCGTCGTATTGCAAGAGTCAGTATGGAGG TGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCG ACAATCACGGCGGAGGATCCGATATCCAGCTGACCCAGAGCCCTAGCAGC CTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCGA GAGCGTGGACGCTTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAAGC CCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGC GGCGTGCCAAGCAGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCT GACCATCAGCAGCATGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGC AGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAAGCTGGAAATC AAG [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD007 2001 (SEQ ID NO: 943)] (SEQ ID NO: 239) [QGQSGQG][VGPCELTPKPVCNTYGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD007 2001 Amino Acid Sequence: (SEQ ID NO: 1052) VGPCELTPKPVCNTYGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD007 2001 (SEQ ID NO: 944)] (SEQ ID NO: 240) [CAAGGCCAGTCTGGCCAAGGT][GTGGGGCCTTGCGAGTTGACGCCGAA GCCTGTTTGCAATACGTATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD008 2001 (SEQ ID NO: 945)] (SEQ ID NO: 241) [QGQSGQG][ETCNQYERSSGLCFAGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD008 2001 Amino Acid Sequence: (SEQ ID NO: 1053) ETCNQYERSSGLCFAGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD008 2001 (SEQ ID NO: 946)] (SEQ ID NO: 242) [CAAGGCCAGTCTGGCCAAGGT][GAGACTTGCAATCAGTATGAGAGGTC GAGTGGTTTGTGCTTTGCGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD009 2001 (SEQ ID NO: 947)] (SEQ ID NO: 243) [QGQSGQG][APRTCYTYQCSSFYTGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD009 2001 Amino Acid Sequence: (SEQ ID NO: 1054) APRTCYTYQCSSFYTGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD009 2001 (SEQ ID NO: 948)] (SEQ ID NO: 244) [CAAGGCCAGTCTGGCCAAGGT][GCGCCGCGGACGTGCTATACGTATCA GTGCTCTAGTTTTTATACTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD010 2001 (SEQ ID NO: 949)] (SEQ ID NO: 245) [QGQSGQG][GLCSWYLSSSGLCVDGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD010 2001 Amino Acid Sequence: (SEQ ID NO: 1055) GLCSWYLSSSGLCVDGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD010 2001 (SEQ ID NO: 950)] (SEQ ID NO: 246) [CAAGGCCAGTCTGGCCAAGGT][GGTCTTTGCAGTTGGTATCTTAGTAG TTCGGGTTTGTGCGTGGATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD011 2001 (SEQ ID NO: 951)] (SEQ ID NO: 247) [QGQSGQG][VPWCQLTPRVMCMWAGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD011 2001 Amino Acid Sequence: (SEQ ID NO: 1056) VPWCQLTPRVMCMWAGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD011 2001 (SEQ ID NO: 952)] (SEQ ID NO: 248) [CAAGGCCAGTCTGGCCAAGGT][GTGCCTTGGTGCCAGTTGACGCCGCG GGTTATGTGCATGTGGGCGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD012 2001 (SEQ ID NO: 953)] (SEQ ID NO: 249) [QGQSGQG][NWLDCQFYSECSVYGGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD012 2001 Amino Acid Sequence: (SEQ ID NO: 953) NWLDCQFYSECSVYGGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD012 2001 (SEQ ID NO: 954)] (SEQ ID NO: 250) [CAAGGCCAGTCTGGCCAAGGT][AATTGGTTGGATTGCCAGTTTTATTC TGAGTGCTCTGTTTATGGTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] PD-1 1.5 PD012 2001 Nucleic Acid Sequence: (SEQ ID NO: 954) AATTGGTTGGATTGCCAGTTTTATTCTGAGTGCTCTGTTTATGGTGGAGG TGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCG ACAATCACGGCGGAGGATCCGATATCCAGCTGACCCAGAGCCCTAGCAGC CTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCGA GAGCGTGGACGCTTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAAGC CCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGC GGCGTGCCAAGCAGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCT GACCATCAGCAGCATGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGC AGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAAGCTGGAAATC AAG [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD013 2001 (SEQ ID NO: 955)] (SEQ ID NO: 251) [QGQSGQG][SCPLYVMSSFGGCWDGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD013 2001 Amino Acid Sequence: (SEQ ID NO: 1057) SCPLYVMSSFGGCWDGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD013 2001 (SEQ ID NO: 956)] (SEQ ID NO: 252) [CAAGGCCAGTCTGGCCAAGGT][TCGTGCCCTTTGTATGTGATGTCTAG TTTTGGTGGGTGCTGGGATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD014 2001 (SEQ ID NO: 957)] (SEQ ID NO: 253) [QGQSGQG][MSHCWMFSSSCDGVKGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD014 2001 Amino Acid Sequence: (SEQ ID NO: 1058) MSHCWMFSSSCDGVKGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD014 2001 (SEQ ID NO: 958)] (SEQ ID NO: 254) [CAAGGCCAGTCTGGCCAAGGT][ATGAGTCATTGCTGGATGTTTTCGAG TTCTTGCGATGGGGTGAAGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD015 2001 (SEQ ID NO: 959)] (SEQ ID NO: 255) [QGQSGQG][VSYCTWLIEVTCLRGGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD015 2001 Amino Acid Sequence: (SEQ ID NO: 1059) VSYCTWLIEVTCLRGGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD015 2001 (SEQ ID NO: 960)] (SEQ ID NO: 256) [CAAGGCCAGTCTGGCCAAGGT][GTTTCGTATTGCACGTGGTTGATTGA GGTGACTTGCCTGAGGGGTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD016 2001 (SEQ ID NO: 961)] (SEQ ID NO: 257) [QGQSGQG][VLCAAYALSSGICGGGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD016 2001 Amino Acid Sequence: (SEQ ID NO: 1060) VLCAAYALSSGICGGGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD016 2001 (SEQ ID NO: 962)] (SEQ ID NO: 258) [CAAGGCCAGTCTGGCCAAGGT][GTTTTGTGCGCTGCTTATGCTTTGAG TTCGGGTATTTGCGGTGGGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD017 2001 (SEQ ID NO: 963)] (SEQ ID NO: 259) [QGQSGQG][TTCNLYQQSSMFCNAGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD017 2001 Amino Acid Sequence: (SEQ ID NO: 1061) TTCNLYQQSSMFCNAGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD017 2001 (SEQ ID NO: 964)] (SEQ ID NO: 260) [CAAGGCCAGTCTGGCCAAGGT][ACGACTTGCAATCTGTATCAGCAGTC TTCTATGTTTTGCAATGCTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD018 2001 (SEQ ID NO: 965)] (SEQ ID NO: 261) [QGQSGQG][APRCYMFASYCKSQYGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD018 2001 Amino Acid Sequence: (SEQ ID NO: 1062) APRCYMFASYCKSQYGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K] [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD018 2001 (SEQ ID NO: 966)] (SEQ ID NO: 262) [CAAGGCCAGTCTGGCCAAGGT][GCGCCTAGGTGCTATATGTTTGCGTC GTATTGCAAGAGTCAGTATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD019 2001 (SEQ ID NO: 967)] (SEQ ID NO: 263) [PD-1 1.5 PD019 2001 (SEQ ID NO: 967)] [QGQSGQG][PCDQNPYFYPYVCHAGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD019 2001 Amino Acid Sequence: (SEQ ID NO: 967) PCDQNPYFYPYVCHAGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD019 2001 (SEQ ID NO: 968)] (SEQ ID NO: 264) [CAAGGCCAGTCTGGCCAAGGT][CCTTGCGATCAGAATCCGTATTTTTA TCCGTATGTGTGCCATGCGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] PD-1 1.5 PD019 2001 Nucleic Acid Sequence: (SEQ ID NO: 968) CCTTGCGATCAGAATCCGTATTTTTATCCGTATGTGTGCCATGCGGGAGG TGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCG ACAATCACGGCGGAGGATCCGATATCCAGCTGACCCAGAGCCCTAGCAGC CTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCGA GAGCGTGGACGCTTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAAGC CCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGC GGCGTGCCAAGCAGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCT GACCATCAGCAGCATGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGC AGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAAGCTGGAAATC AAG [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD020 2001 (SEQ ID NO: 969)] (SEQ ID NO: 265) [QGQSGQG][SVCPMYALSSMLCGAGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD020 2001 Amino Acid Sequence: (SEQ ID NO: 1063) SVCPMYALSSMLCGAGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD020 2001 (SEQ ID NO: 970)] (SEQ ID NO: 266) [CAAGGCCAGTCTGGCCAAGGT][TCTGTGTGCCCTATGTATGCGTTGAG TTCTATGTTGTGCGGTGCGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD021 2001 (SEQ ID NO: 971)] (SEQ ID NO: 267) [QGQSGQG][LSVECYVFSRCSSLPGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD021 2001 Amino Acid Sequence: (SEQ ID NO: 1064) LSVECYVFSRCSSLPGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K] [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD021 2001 (SEQ ID NO: 972)] (SEQ ID NO: 268) [CAAGGCCAGTCTGGCCAAGGT][TTGTCTGTGGAGTGCTATGTGTTTTC GCGGTGCAGTAGTCTGCCGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD022 2001 (SEQ ID NO: 973)] (SEQ ID NO: 269) [QGQSGQG][FYCTYLVSLTCHPQGGGSSGGSISSGLLSGRSDNHGGGSD IQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLL IYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWT FGQGTKLEIK] PD-1 1.5 PD022 2001 Amino Acid Sequence: (SEQ ID NO: 1065) FYCTYLVSLTCHPQGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSSL SASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSG VPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD022 2001 (SEQ ID NO: 974)] (SEQ ID NO: 270) [CAAGGCCAGTCTGGCCAAGGT][TTTTATTGCACTTATTTGGTGTCTTT GACTTGCCATCCGCAGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCG GACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATCCAG CTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGAC CATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCTTCA TGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTAC GCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTCTGG CAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGGACT TCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTTGGC CAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD023 2001 (SEQ ID NO: 975)] (SEQ ID NO: 271) [QGQSGQG][SMAGCQWSSFCVQRDGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD023 2001 Amino Acid Sequence: (SEQ ID NO: 1066) SMAGCQWSSFCVQRDGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD023 2001 (SEQ ID NO: 976)] (SEQ ID NO: 272) [CAAGGCCAGTCTGGCCAAGGT][TCTATGGCGGGTTGCCAGTGGAGTTC GTTTTGCGTGCAGCGGGATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD024 2001 (SEQ ID NO: 977)] (SEQ ID NO: 273) [QGQSGQG][IYSCYMFASRCTSDKGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD024 2001 Amino Acid Sequence: (SEQ ID NO: 1067) IYSCYMFASRCTSDKGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K] [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD024 2001 (SEQ ID NO: 978)] (SEQ ID NO: 274) [CAAGGCCAGTCTGGCCAAGGT][ATTTATTCGTGCTATATGTTTGCTTC GCGGTGCACGTCTGATAAGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD002 3001 (SEQ ID NO: 979)] (SEQ ID NO: 275) [QGQSGQG][DVNCAIWYSVCTTVPGGGSSGGAVGLLAPPGGLSGRSDNH GGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKA PKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKD VPWTFGQGTKLEIK] PD-1 1.5 PD002 3001 Amino Acid Sequence: (SEQ ID NO: 1068) DVNCAIWYSVCTTVPGGGSSGGAVGLLAPPGGLSGRSDNHGGSDIQLTQS PSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASN QGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTK LEIK [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD002 3001 (SEQ ID NO: 980)] (SEQ ID NO: 276) [CAAGGCCAGTCTGGCCAAGGT]CAAGGCCAGTCTGGCCAAGGTGATGTT AATTGCGCTATTTGGTATTCGGTGTGCACTACTGTTCCTGGAGGTGGCTC GAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATC ACGGCGGAGGATCCGGAGGTGGCTCGAGCGGCGGCGCTGTGGGACTGCTG GCTCCTCCTGGTGGCCTGTCTGGCAGATCTGATAACCACGGAGGATCCGA TATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACA GAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATC AGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCT GATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCG GCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCC GAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGAC CTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD-12 3001 (SEQ ID NO: 981)] (SEQ ID NO: 277) [QGQSGQG][NWLDCQFYSECSVYGGGGSSGGAVGLLAPPGGLSGRSDNH GGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKA PKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKD VPWTFGQGTKLEIK] PD-1 1.5 PD-12 3001 Amino Acid Sequence: (SEQ ID NO: 1069) NWLDCQFYSECSVYGGGGSSGGAVGLLAPPGGLSGRSDNHGGSDIQLTQS PSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASN QGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTK LEIK] [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD-12 3001 (SEQ ID NO: 982)] (SEQ ID NO: 278) [CAAGGCCAGTCTGGCCAAGGT][AATTGGTTGGATTGCCAGTTTTATTC TGAGTGCTCTGTTTATGGTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGGAGGT GGCTCGAGCGGCGGCGCTGTGGGACTGCTGGCTCCTCCTGGTGGCCTGTC TGGCAGATCTGATAACCACGGAGGATCCGATATCCAGCTGACCCAGAGCC CTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCGAGAGCGTGGACGCTTACGGCATCAGCTTCATGAACTGGTTCCA GCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATC AGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTCTGGCAGCGGCACCGAC TTCACCCTGACCATCAGCAGCATGCAGCCCGAGGACTTCGCCACCTACTA CTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAAGC TGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD-16 3001 (SEQ ID NO: 983)] (SEQ ID NO: 279) [QGQSGQG][VLCAAYALSSGICGGGGGSSGGAVGLLAPPGGLSGRSDNH GGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKA PKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKD VPWTFGQGTKLEIK] PD-1 1.5 PD-16 3001 Amino Acid Sequence: (SEQ ID NO: 1070) VLCAAYALSSGICGGGGGSSGGAVGLLAPPGGLSGRSDNHGGSDIQLTQS PSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASN QGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTK LEIK [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD-16 3001 (SEQ ID NO: 984)] (SEQ ID NO: 280) [CAAGGCCAGTCTGGCCAAGGT][GTTTTGTGCGCTGCTTATGCTTTGAG TTCGGGTATTTGCGGTGGGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGGAGGT GGCTCGAGCGGCGGCGCTGTGGGACTGCTGGCTCCTCCTGGTGGCCTGTC TGGCAGATCTGATAACCACGGAGGATCCGATATCCAGCTGACCCAGAGCC CTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACCTGTAGA GCCAGCGAGAGCGTGGACGCTTACGGCATCAGCTTCATGAACTGGTTCCA GCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATC AGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTCTGGCAGCGGCACCGAC TTCACCCTGACCATCAGCAGCATGCAGCCCGAGGACTTCGCCACCTACTA CTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAAGC TGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD025 2001 (SEQ ID NO: 985)] (SEQ ID NO: 281) [QGQSGQG][SRCSVYEVSSGLCDWGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD025 2001 Amino Acid Sequence: (SEQ ID NO: 1071) SRCSVYEVSSGLCDWGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD025 2001 (SEQ ID NO: 986)] (SEQ ID NO: 282) [CAAGGCCAGTCTGGCCAAGGT][TCTCGTTGCTCTGTGTATGAGGTTTC GTCGGGGCTGTGCGATTGGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD026 2001 (SEQ ID NO: 987)] (SEQ ID NO: 283) [QGQSGQG][GMCSAYAYSSKLCTIGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD026 2001 Amino Acid Sequence: (SEQ ID NO: 1072) GMCSAYAYSSKLCTIGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD026 2001 (SEQ ID NO: 988)] (SEQ ID NO: 284) [CAAGGCCAGTCTGGCCAAGGT][GGGATGTGCTCGGCGTATGCTTATTC GAGTAAGTTGTGCACTATTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD027 2001 (SEQ ID NO: 989)] (SEQ ID NO: 285) [QGQSGQG][MTTNTCNLLCQQFLTGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD027 2001 Amino Acid Sequence: (SEQ ID NO: 1073) MTTNTCNLLCQQFLTGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K] [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD027 2001 (SEQ ID NO: 990)] (SEQ ID NO: 286) [CAAGGCCAGTCTGGCCAAGGT][ATGACTACGAATACTTGCAATCTGTT GTGCCAGCAGTTTTTGACGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD028 2001 (SEQ ID NO: 991)] (SEQ ID NO: 287) [QGQSGQG][FQPCLMFASSCFTSKGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD028 2001 Amino Acid Sequence: (SEQ ID NO: 991) FQPCLMFASSCFTSKGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD028 2001 (SEQ ID NO: 992)] (SEQ ID NO: 288) [CAAGGCCAGTCTGGCCAAGGT][TTTCAGCCGTGCCTGATGTTTGCGAG TAGTTGCTTTACTAGTAAGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] PD-1 1.5 PD028 2001 Nucleic Acid Sequence: (SEQ ID NO: 992) TTTCAGCCGTGCCTGATGTTTGCGAGTAGTTGCTTTACTAGTAAGGGAGG TGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCG ACAATCACGGCGGAGGATCCGATATCCAGCTGACCCAGAGCCCTAGCAGC CTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCGA GAGCGTGGACGCTTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAAGC CCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGC GGCGTGCCAAGCAGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCT GACCATCAGCAGCATGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGC AGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAAGCTGGAAATC AAG [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD030 2001 (SEQ ID NO: 993)] (SEQ ID NO: 289) [QGQSGQG][ALCSMYLASSGLCNKGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD030 2001 Amino Acid Sequence: (SEQ ID NO: 1074) ALCSMYLASSGLCNKGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD030 2001 (SEQ ID NO: 994)] (SEQ ID NO: 290) [CAAGGCCAGTCTGGCCAAGGT][GCGCTTTGCAGTATGTATCTTGCTAG TTCTGGGCTGTGCAATAAGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD033 2001 (SEQ ID NO: 995)] (SEQ ID NO: 291) [QGQSGQG][EFCARDWLPYQCSSFGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD-1 1.5 PD033 2001 Amino Acid Sequence: (SEQ ID NO: 1075) EFCARDWLPYQCSSFGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K] [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD033 2001 (SEQ ID NO: 996)] (SEQ ID NO: 292) [CAAGGCCAGTCTGGCCAAGGT][GAGTTTTGCGCTCGGGATTGGCTGCC GTATCAGTGCTCGAGTTTTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD034 2001 (SEQ ID NO: 1028)] (SEQ ID NO: 1029) [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDNHG GGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQ KPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYY CQQSKDVPWTFGQGTKLEIK] PD-1 1.5 PD034 2001 Amino Acid Sequence: (SEQ ID NO: 1028) TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPS SLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQ GSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTK LEIK [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD034 2001 (SEQ ID NO: 1030) (SEQ ID NO: 1031) [CAAGGCCAGTCTGGCCAAGGT][ACGTCATACTGCAGTATTGAGCAT TACCCCTGCAATACACATCATGGAGGTGGCTCGAGCGGCGGCTCTATCTC TTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATA TCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAG AGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATC AGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGC TGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTC CGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAG CCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCC TGGACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] PD-1 1.5 PD034 2001 Nucleic Acid Sequence: (SEQ ID NO: 1030) ACGTCATACTGCAGTATTGAGCATTACCCCTGCAATACACATCATGGAG GTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATC CGACAATCACGGCGGAGGATCCGATATCCAGCTGACCCAGAGCCCTAGC AGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCA GCGAGAGCGTGGACGCTTACGGCATCAGCTTCATGAACTGGTTCCAGCA GAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAATCAG GGCAGCGGCGTGCCAAGCAGATTTTCCGGCTCTGGCAGCGGCACCGACT TCACCCTGACCATCAGCAGCATGCAGCCCGAGGACTTCGCCACCTACTA CTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTTGGCCAGGGTACCAAG CTGGAAATCAAG [Spacer (SEQ ID NO: 362)]  [PD-1 1.5 PD035 2001 (SEQ ID NO: 997)] (SEQ ID NO: 293) [QGQSGQG][PYICSSFPLDCQAGQGGGSSGGSISSGLLSGRSDNHGG GSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAP KLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDV PWTFGQGTKLEIK] PD-1 1.5 PD035 2001 Amino Acid Sequence: (SEQ ID NO: 1076) PYICSSFPLDCQAGQGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPS SLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQ GSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTK LEIK] [Spacer (SEQ ID NO: 918)]  [PD-1 1.5 PD035 2001 (SEQ ID NO: 998)] (SEQ ID NO: 584) [CAAGGCCAGTCTGGCCAAGGT][CCTTATATTTGCTCTAGTTTTCCGTT GGATTGCCAGGCGGGTCAGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAG TGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAG CTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG ATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCG GCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCC CGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGG ACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG]

Example 9: Anti-PD-1 Activatable Antibodies of the Disclosure

This example demonstrates that anti-PD-1 activatable antibodies of the disclosure can be made in a variety of combinations of MM, CM, VL, and VH domains, as well as in a variety of distinct Ig isotypes. In addition, this example demonstrates that anti-PD-1 activatable antibodies of the disclosure can be made in a variety of combinations of MM, CM, VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2, and VHCDR3 domains, as well as in a variety of distinct Ig isotypes.

TABLE 13A VL VL CDRs VH CDRs SEQ VH SEQ ID NO: SEQ ID NO: Mask Sequence Substrates: ID NO: SEQ ID NO: CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 AMSGCSWSAFCPYLA LSGRSDNH 1 3 653 658 664 669 678 683 (SEQ ID NO: 66) (SEQ ID NO: 294) DVNCAIWYSVCITVP TGRGPSWV 5 7 654 659 665 670 679 684 (SEQ ID NO: 67) (SEQ ID NO: 295) LVCPLYALSSGVCMG PLTGRSGG 9 11 655 660 666 671 680 685 (SEQ ID NO: 68) (SEQ ID NO: 296) SVNCRIWSAVCAGYE TARGPSFK 13 15 656 661 667 672 681 686 (SEQ ID NO: 69) (SEQ ID NO: 297) MLVCSLQPTAMCERV NTLSGRSENHSG 17 19 657 662 668 673 682 687 (SEQ ID NO: 70) (SEQ ID NO: 298) APRCYMFASYCKSQY NTLSGRSGNHGS 21 39 663 674 (SEQ ID NO: 71) (SEQ ID NO: 299) VGPCELTPKPVCNTY TSTSGRSANPRG 23 41 675 (SEQ ID NO: 72) (SEQ ID NO: 300) ETCNQYERSSGLCFA TSGRSANP 25 43 676 (SEQ ID NO: 73) (SEQ ID NO: 301) APRTCYTYQCSSFYT VHMPLGFLGP 27 45 677 (SEQ ID NO: 74) (SEQ ID NO: 302) GLCSWYLSSSGLCVD AVGLLAPP 29 47 VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 75) (SEQ ID NO: 303) NO: 1 NO: 3 VPWCQLTPRVMCMWA AQNLLGMV 31 49 VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 76) (SEQ ID NO: 304) NO: 5 NO: 7 NWLDCQFYSECSVYG QNQALRMA 33 51 VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 77) (SEQ ID NO: 305) NO: 9 NO: 11 SCPLYVMSSFGGCWD LAAPLGLL 35 53 VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 78) (SEQ ID NO: 306) NO: 13 NO: 15 MSHCWMFSSSCDGVK STFPFGMF 37 55 VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 79) (SEQ ID NO: 307) NO: 17 NO: 19 VSYCTWLIEVICLRG ISSGLLSS 57 VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 80) (SEQ ID NO: 308) NO: 21 NO: 39 VLCAAYALSSGICGG PAGLWLDP 59 VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 81) (SEQ ID NO: 309) NO: 23 NO: 41 TTCNLYQQSSMFCNA VAGRSMRP VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 82) (SEQ ID NO: 310) NO: 25 NO: 43 APRCYMFASYCKSQY VVPEGRRS VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 83) (SEQ ID NO: 311) NO: 27 NO: 45 PCDQNPYFYPYVCHA ILPRSPAF VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 84) (SEQ ID NO: 312) NO: 29 NO: 47 SVCPMYALSSMLCGA MVLGRSLL VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 85) (SEQ ID NO: 313) NO: 31 NO: 49 LSVECYVFSRCSSLP QGRAITFI VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 86) (SEQ ID NO: 314) NO: 33 NO: 51 FYCTYLVSLTCHPQ SPRSIMLA VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 87) (SEQ ID NO: 315) NO: 35 NO: 53 SMAGCQWSSFCVQRD SMLRSMPL VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 88) (SEQ ID NO: 316) NO: 37 NO: 55 IYSCYMFASRCTSDK SARGPSRW VH CDRs of SEQ ID (SEQ ID NO: 89) (SEQ ID NO: 319) NO: 57 SRCSVYEVSSGLCDW GWHTGRN VH CDRs of SEQ ID (SEQ ID NO: 90) (SEQ ID NO: 320) NO: 59 GMCSAYAYSSKLCTI HTGRSGAL LC CDRs of SEQ ID HC CDRs of SEQ ID (SEQ ID NO: 91) (SEQ ID NO: 321) NO: 543 NO: 546 MTTNTCNLLCQQFLT AARGPAIH (SEQ ID NO: 92) (SEQ ID NO: 322) FQPCLMFASSCFTSK RGPAFNPM (SEQ ID NO: 93) (SEQ ID NO: 323) WNCHPAGVGPVFCEV SSRGPAYL (SEQ ID NO: 94) (SEQ ID NO: 324) ALCSMYLASSGLCNK RGPATPIM (SEQ ID NO: 95) (SEQ ID NO: 325) NYLSCQFFQNCYETY RGPA (SEQ ID NO: 96) (SEQ ID NO: 326) GWCLFSDMWLGLCSA GGQPSGMWGW (SEQ ID NO: 97) (SEQ ID NO: 327) EFCARDWLPYQCSSF FPRPLGITGL (SEQ ID NO: 98) (SEQ ID NO: 328) TSYCSIEHYPCNTHH SPLTGRSG (SEQ ID NO: 99) (SEQ ID NO: 329) PYICSSFPLDCQAGQ SAGFSLPA (SEQ ID NO: 100) (SEQ ID NO: 330) VGCEWYMSSSGMCSR LAPLGLQRR (SEQ ID NO: 101) (SEQ ID NO: 331) EVCGGCSMQSVSCWP SGGPLGVR (SEQ ID NO: 102) (SEQ ID NO: 332) FTECQLSPKAICMSN PLGL (SEQ ID NO: 103) (SEQ ID NO: 333) KYCLFSEYVEGTCLN ISSGLSS (SEQ ID NO: 104) (SEQ ID NO: 334) SGCPMYAWGWDECWR PVGYTSSL (SEQ ID NO: 105) (SEQ ID NO: 335) VDCPWYASSSAICSR DWLYWPGI (SEQ ID NO: 106) (SEQ ID NO: 336) DMLLCQIRGSCAAWG LKAAPRWA (SEQ ID NO: 107) (SEQ ID NO: 337) ECHPYQASASLWCGY GPSHLVLT (SEQ ID NO: 108) (SEQ ID NO: 338) MMMGCMWSAWCPPSR LPGGLSPW (SEQ ID NO: 109) (SEQ ID NO: 339) NAYFRCSLMCNMIMF MGLFSEAG (SEQ ID NO: 110) (SEQ ID NO: 340) ACCKESVHSVHDCKR SPLPLRVP (SEQ ID NO: 111) (SEQ ID NO: 341) ACIGINSYMSNYCYL RMHLRSLG (SEQ ID NO: 112) (SEQ ID NO: 342) ANCSFLELTNKFCTI LLAPSHRA (SEQ ID NO: 113) (SEQ ID NO: 343) AYCSYLMFASNPCII GPRSFGL (SEQ ID NO: 114) (SEQ ID NO: 344) CFTSKCPCLCYSLLA GPRSFG (SEQ ID NO: 115) (SEQ ID NO: 345) CLCRDINCWLGCSKT LSGRSGNH (SEQ ID NO: 116) (SEQ ID NO: 1157) CWCDIYCSPYQCSSF SGRSANPRG (SEQ ID NO: 117) (SEQ ID NO: 1158) DCIYYYQQSANLCSY LSGRSDDH (SEQ ID NO: 118) (SEQ ID NO: 1161) DCTGVNYYIDKHCIN LSGRSDIH (SEQ ID NO: 119) (SEQ ID NO: 1162) DECHGYLRSSGLCGG LSGRSDQH (SEQ ID NO: 120) (SEQ ID NO: 1165) DICSAYAASSGFCYY LSGRSDTH (SEQ ID NO: 121) (SEQ ID NO: 1166) DIICVLIPTAWCGRT LSGRSDYH (SEQ ID NO: 122) (SEQ ID NO: 1169) DNCCMYCSWWIACRD LSGRSDNP (SEQ ID NO: 123) (SEQ ID NO: 1520) DSCQWYMLSADLCGT LSGRSANP (SEQ ID NO: 124) (SEQ ID NO: 1695) DSVCFSSSSFLCHKS LSGRSANI (SEQ ID NO: 125) (SEQ ID NO: 1696) DTMCAIWWTVCSGGR LSGRSDNI (SEQ ID NO: 126) (SEQ ID NO: 1697) ECTYQTSSFHEACMS MIAPVAYR (SEQ ID NO: 127) (SEQ ID NO: 1698) EGCNLYERSSYGCNN RPSPMWAY (SEQ ID NO: 128) (SEQ ID NO: 1699) EGCTAFAMSAGICGG WATPRPMR (SEQ ID NO: 129) (SEQ ID NO: 1700) EQSCSLTPIAFCWSE FRLLDWQW (SEQ ID NO: 130) (SEQ ID NO: 1701) EWCNAYISSSKLCST ISSGL (SEQ ID NO: 131) (SEQ ID NO: 1702) FEVCYMFASACRNGM ISSGLLS (SEQ ID NO: 132) (SEQ ID NO: 1703) FSCSWYAESSSLCDI ISSGLL (SEQ ID NO: 133) (SEQ ID NO: 1704) FVCQMFEASSGLCGG ISSGLLSGRSDNH (SEQ ID NO: 134) (SEQ ID NO: 214) FYCPCCMFASSCGSR AVGLLAPPGGLSGRSDNH (SEQ ID NO: 135) (SEQ ID NO: 318) FYCSYLPGASHQCSH ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 136) (SEQ ID NO: 346) FYCSYLYMCEVCCYE ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 137) (SEQ ID NO: 346) GFCTQHTVLTWCPTS AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 138) (SEQ ID NO: 347) GSCPSYAVSAGLCYA TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 139) (SEQ ID NO: 348) GSQCFLTPTAFCTHT VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 140) (SEQ ID NO: 349) GTCHPYMQSSKICNN TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 141) (SEQ ID NO: 350) GVECFVFTGGCGGYG LSGRSDNHGGAVGLLAPP (SEQ ID NO: 142) (SEQ ID NO: 351) HELCNGHWVPCCWAY VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 143) (SEQ ID NO: 352) ICDSYYAVSSGLCLL LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 144) (SEQ ID NO: 353) IGCAWYVSSAGWCSP LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 145) (SEQ ID NO: 354) INLCWMFASECGEHH LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 146) (SEQ ID NO: 355) KCWLAEMTNLEHCNM ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 147) (SEQ ID NO: 356) KHCSDFAYSSRLCDR LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 148) (SEQ ID NO: 357) KVCSSYASSSGLCGW QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 149) (SEQ ID NO: 358) LDS CYMFASYCVQAV LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 150) (SEQ ID NO: 359) LLACHPIFVTVCQTR QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 151) (SEQ ID NO: 360) LLSCPYNPEHVCHTS ISSGLLSGRSGNH (SEQ ID NO: 152) (SEQ ID NO: 361) LMCSLYALSSNLCGR ISSGLLSSGGSGGSLSGRNH (SEQ ID NO: 153) (SEQ ID NO: 1091) LMWCVLFLWSWCCRI ISSGLLSGRSANPRG (SEQ ID NO: 154) (SEQ ID NO: 1092) LPI CHLTPTAVCTHI AVGLLAPPTSGRSANPRG (SEQ ID NO: 155) (SEQ ID NO: 1093) LSNMCLAFGSCLYAW AVGLLAPPSGRSANPRG (SEQ ID NO: 156) (SEQ ID NO: 1094) LSRCHPIWYTICQNP ISSGLLSGRSDDH (SEQ ID NO: 157) (SEQ ID NO: 1095) LTQCMSVHKECGGYE ISSGLLSGRSDIH (SEQ ID NO: 158) (SEQ ID NO: 1096) LVNCRIWSWVCEEAT ISSGLLSGRSDQH (SEQ ID NO: 159) (SEQ ID NO: 1097) LYCSWYQMSSAVCKE ISSGLLSGRSDTH (SEQ ID NO: 160) (SEQ ID NO: 1098) MECGWYALSARFCEV ISSGLLSGRSDYH (SEQ ID NO: 161) (SEQ ID NO: 1099) MTCSPYAMSAHFCNE ISSGLLSGRSDNP (SEQ ID NO: 162) (SEQ ID NO: 1100) MVCSLYAYSASLCGA ISSGLLSGRSANP (SEQ ID NO: 163) (SEQ ID NO: 1101) NALCWSTFSWWCDMD ISSGLLSGRSANI (SEQ ID NO: 164) (SEQ ID NO: 1102) NFTCMLTPKAYCVQT AVGLLAPPGGLSGRSDDH (SEQ ID NO: 165) (SEQ ID NO: 1103) NGACIFILSWCINKT AVGLLAPPGGLSGRSDIH (SEQ ID NO: 166) (SEQ ID NO: 1104) NGCELYAAASGLCRT AVGLLAPPGGLSGRSDQH (SEQ ID NO: 167) (SEQ ID NO: 1105) NIECSVFGRCCCDNY AVGLLAPPGGLSGRSDTH (SEQ ID NO: 168) (SEQ ID NO: 1106) PACRPMFWNRSCDNI AVGLLAPPGGLSGRSDYH (SEQ ID NO: 169) (SEQ ID NO: 1107) PCRVSNMFFPYNCLD AVGLLAPPGGLSGRSDNP (SEQ ID NO: 170) (SEQ ID NO: 1108) PIMCMLLPESYCWIW AVGLLAPPGGLSGRSANP (SEQ ID NO: 171) (SEQ ID NO: 1109) PQSCYMFASLCMPNG AVGLLAPPGGLSGRSANI (SEQ ID NO: 172) (SEQ ID NO: 1110) PRCPQGLPLYQCSSF ISSGLLSGRSDNI (SEQ ID NO: 173) (SEQ ID NO: 1111) PSVECLVFKRCYALP AVGLLAPPGGLSGRSDNI (SEQ ID NO: 174) (SEQ ID NO: 1112) PVCQRSATIYNCNWF (SEQ ID NO: 175) QCAAYYISSFGGCSN (SEQ ID NO: 176) QFGCFMLARDFCGTY (SEQ ID NO: 177) QMMCPYNPEHKCHQK (SEQ ID NO: 178) QRECWMFASSCNSKN (SEQ ID NO: 179) QSNMCITYICSSFNY (SEQ ID NO: 180) QSRCHSLAPYLCSSF (SEQ ID NO: 181) RAYCSLLFADSCNNN (SEQ ID NO: 182) RCIGINQYIDSNCYN (SEQ ID NO: 183) RLSCFMFASQCALEF (SEQ ID NO: 184) RQCIILMNHRQCFFK (SEQ ID NO: 185) RSCTPYMMSSSLCNT (SEQ ID NO: 186) RYCHYWKMPYECSSF (SEQ ID NO: 187) SCVSLSWFDMLKCYE (SEQ ID NO: 188) SDNCEIWWTVCSAAM (SEQ ID NO: 189) SFCWSYLVSSGLCGV (SEQ ID NO: 190) SMCMNNYGTTIMCGN (SEQ ID NO: 191) SMVGCGWSTFCPSRG (SEQ ID NO: 192) SSLHCANGHTCPFCL (SEQ ID NO: 193) SVCSYYEESSGICSP (SEQ ID NO: 194) SWCGWYAASSGVCAL (SEQ ID NO: 195) TCISQTIDSYLNCVN (SEQ ID NO: 196) TFCNLYTKSSNICMS (SEQ ID NO: 197) TYCVFHEYLDNTCNN (SEQ ID NO: 198) VATGCPNLMLCGSWP (SEQ ID NO: 199) VEYCSLLLGNRCDYW (SEQ ID NO: 200) VGCNMYLMSAGLCVD (SEQ ID NO: 201) VLYCSWDSGTCVGSH (SEQ ID NO: 202) VMFSCYYLETCAPGV (SEQ ID NO: 203) VRIGLCPESCLVSGF (SEQ ID NO: 204) VTCTYYATSSSLCNT (SEQ ID NO: 205) VTGCILLPKAWCWGD (SEQ ID NO: 206) VWCSIYEYSSNLCSR (SEQ ID NO: 207) WMLECQYNNTCNNMT (SEQ ID NO: 208) WPCSPLEYYNNICNV (SEQ ID NO: 209) WTYDCHLNQTCPTYY (SEQ ID NO: 210) YCSINMYLIGGNCMY (SEQ ID NO: 211) YFCSLYANSAGFCGG (SEQ ID NO: 212) YVSCYMFSSSCPSTW (SEQ ID NO: 213)

TABLE 13B VL VH VL CDRs VH CDRs SEQ SEQ SEQ ID NO: SEQ ID NO: Mask Sequence Substrates: ID NO: ID NO: CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 RYCHAANPDRFCGIY LSGRSDNH 626 1346 VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 1206) (SEQ ID NO: 294) NO: 626 NO: 1346 PRVCSTDGGDYCLLP TGRGPSWV 1705 1706 1707 1708 1709 1710 (SEQ ID NO: 1207) (SEQ ID NO: 295) PRPQCHHRHNCPDHP PLTGRSGG (SEQ ID NO: 1208) (SEQ ID NO: 296) KCSRPAHQNPDRCSR TARGPSFK (SEQ ID NO: 1209) (SEQ ID NO: 297) ASYRCPDYKCSHTKH NTLSGRSENHSG (SEQ ID NO: 1210) (SEQ ID NO: 298) LPRCPDHPIKCIETK NTLSGRSGNHGS (SEQ ID NO: 1211) (SEQ ID NO: 299) YTFGCPDRYCDRAAT TSTSGRSANPRG (SEQ ID NO: 1212) (SEQ ID NO: 300) RGCPDFNPPSHCYTA TSGRSANP (SEQ ID NO: 1213) (SEQ ID NO: 301) RDYCGPQSPDYCHEI VHMPLGFLGP (SEQ ID NO: 1214) (SEQ ID NO: 302) PNKPCPDLQCYVTNY AVGLLAPP (SEQ ID NO: 1215) (SEQ ID NO: 303) PRVACGEPDLCYSNT AQNLLGMV (SEQ ID NO: 1216) (SEQ ID NO: 304) RGCKKHTISTLTCPD QNQALRMA (SEQ ID NO: 1217) (SEQ ID NO: 305) PAYRCPDRPPCKNQM LAAPLGLL (SEQ ID NO: 1218) (SEQ ID NO: 306) NARCYPYFGDNCHMN STFPFGMF (SEQ ID NO: 1220) (SEQ ID NO: 307) PTLRCPDRWCYDSPR ISSGLLSS (SEQ ID NO: 1221) (SEQ ID NO: 308) PSNLCPDKWCQTWRS PAGLWLDP (SEQ ID NO: 1222) (SEQ ID NO: 309) TPRYCAASYCPAHGY VAGRSMRP (SEQ ID NO: 1223) (SEQ ID NO: 310) RPGCGAVSPRCPDAP VVPEGRRS (SEQ ID NO: 1224) (SEQ ID NO: 311) VLRCHKQNPDNCNNH ILPRSPAF (SEQ ID NO: 1225) (SEQ ID NO: 312) GVKSCREPDFCSRGS MVLGRSLL (SEQ ID NO: 1226) (SEQ ID NO: 313) RNNLCPDYSCNNHNS QGRAITFI (SEQ ID NO: 1227) (SEQ ID NO: 314) RAACHRLNPDACTNG SPRSIMLA (SEQ ID NO: 1228) (SEQ ID NO: 315) VCQSDRIPDYVICTD SMLRSMPL (SEQ ID NO: 1229) (SEQ ID NO: 316) RNCRIASINPDYCNI SARGPSRW (SEQ ID NO: 1230) (SEQ ID NO: 319) KEWRCPDYKCKPSYH GWHTGRN (SEQ ID NO: 1231) (SEQ ID NO: 320) NLRICHKSLCPDYIK HTGRSGAL (SEQ ID NO: 1232) (SEQ ID NO: 321) NTHKCSNTNICPSFN AARGPAIH (SEQ ID NO: 1233) (SEQ ID NO: 322) STRYCQASQCQMSPY RGPAFNPM (SEQ ID NO: 1234) (SEQ ID NO: 323) THRFCTASLCNKNTS SSRGPAYL (SEQ ID NO: 1235) (SEQ ID NO: 324) YTLCNTRSPDWCPNK RGPATPIM (SEQ ID NO: 1236) (SEQ ID NO: 325) IRCTTGQSPDYCPQS RGPA (SEQ ID NO: 1237) (SEQ ID NO: 326) RCNQPDKNDQMLCNI GGQPSGMWGW (SEQ ID NO: 1238) (SEQ ID NO: 327) GTCRTDHQSPDYCYY FPRPLGITGL (SEQ ID NO: 1239) (SEQ ID NO: 328) RGCFRSGDSLGMCPD SPLTGRSG (SEQ ID NO: 1240) (SEQ ID NO: 329) SGCFDSNEHRHCSRI SAGFSLPA (SEQ ID NO: 1241) (SEQ ID NO: 330) NRCMKLWYNPDCVAR LAPLGLQRR (SEQ ID NO: 1242) (SEQ ID NO: 331) PLCARPHYWSPCDQS SGGPLGVR (SEQ ID NO: 1243) (SEQ ID NO: 332) DSKCHPNSPDYCFNS PLGL (SEQ ID NO: 1244) (SEQ ID NO: 333) NGSCRPLGGDFCGNR ISSGLSS (SEQ ID NO: 1245) (SEQ ID NO: 334) KTRCIEMSGDYCAKS PVGYTSSL (SEQ ID NO: 1246) (SEQ ID NO: 335) IRPCMYNWGDLCNQF DWLYWPGI (SEQ ID NO: 1247) (SEQ ID NO: 336) VKTCMENNPDYCYNN LKAAPRWA (SEQ ID NO: 1248) (SEQ ID NO: 337) LRMCFEASGDYCDQQ GPSHLVLT (SEQ ID NO: 1249) (SEQ ID NO: 338) IRKCQLDGPDQCMLT LPGGLSPW (SEQ ID NO: 1250) (SEQ ID NO: 339) KWKCHKNNPNYCNNR MGLFSEAG (SEQ ID NO: 1251) (SEQ ID NO: 340) RTMCLDTNPDYCQSH SPLPLRVP (SEQ ID NO: 1252) (SEQ ID NO: 341) LAACHSMDSHRCPDY RMHLRSLG (SEQ ID NO: 1253) (SEQ ID NO: 342) RSPCIHNATMCPDYT LLAPSHRA (SEQ ID NO: 1254) (SEQ ID NO: 343) MPRCPDWPPRCSMVI GPRSFGL (SEQ ID NO: 1255) (SEQ ID NO: 344) VRQLCRLPDYCPSGK GPRSFG (SEQ ID NO: 1256) (SEQ ID NO: 345) PRPPCAQSLNCPDRA LSGRSGNH (SEQ ID NO: 1257) (SEQ ID NO: 1157) SFGRCTLVRTCPDFM SGRSANPRG (SEQ ID NO: 1258) (SEQ ID NO: 1158) RDKPCPDFSCATIHY LSGRSDDH (SEQ ID NO: 1259) (SEQ ID NO: 1161) ATKPCPDRWCTMSTL LSGRSDIH (SEQ ID NO: 1260) (SEQ ID NO: 1162) SSNRCPDLRCTHHNM LSGRSDQH (SEQ ID NO: 1261) (SEQ ID NO: 1165) RGSMCPDLHCSLSHI LSGRSDTH (SEQ ID NO: 1262) (SEQ ID NO: 1166) NYQRCPDRTCMHNII LSGRSDYH (SEQ ID NO: 1263) (SEQ ID NO: 1169) QKRPCPDRKCHAHYN LSGRSDNP (SEQ ID NO: 1264) (SEQ ID NO: 1520) QNHRCPDRWCNKTTN LSGRSANP (SEQ ID NO: 1265) (SEQ ID NO: 1695) RLNLCPDKHCHMTNL LSGRSANI (SEQ ID NO: 1266) (SEQ ID NO: 1696) PQDRCPDKRCTNPGN LSGRSDNI (SEQ ID NO: 1267) (SEQ ID NO: 1697) SRWRCPDYKCEHGKY MIAPVAYR (SEQ ID NO: 1268) (SEQ ID NO: 1698) YENQCPDLYCNRYSM RPSPMWAY (SEQ ID NO: 1269) (SEQ ID NO: 1699) TARSCPVFNCPDNNS WATPRPMR (SEQ ID NO: 1270) (SEQ ID NO: 1700) MDQRCPDAWCTSKPK FRLLDWQW (SEQ ID NO: 1271) (SEQ ID NO: 1701) GDLRCPDRLCPRHSL ISSGL (SEQ ID NO: 1272) (SEQ ID NO: 1702) IQYLCPDYHCKASNN ISSGLLS (SEQ ID NO: 1273) (SEQ ID NO: 1703) QHHRCPDRYCNSNNN ISSGLL (SEQ ID NO: 1274) (SEQ ID NO: 1704) TVALCPDYSCYHINN ISSGLLSGRSDNH (SEQ ID NO: 1275) (SEQ ID NO: 214) SPWRCPDRYCLSNHD AVGLLAPPGGLSGRSDNH (SEQ ID NO: 1276) (SEQ ID NO: 318) SSKRCPDRFCNKTHA ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 1277) (SEQ ID NO: 346) HTDRCPDYKCSQNHF ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 1278) (SEQ ID NO: 346) SRSNCTPQRCNSDYH AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 1279) (SEQ ID NO: 347) FAARCPDKYCAIHTN TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 1280) (SEQ ID NO: 348) GSARCPDLVCQQTKQ VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 1281) (SEQ ID NO: 349) RNLMCPDKFCNKNTK TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 1282) (SEQ ID NO: 350) NIRLCPDKVCTPTWV LSGRSDNHGGAVGLLAPP (SEQ ID NO: 1283) (SEQ ID NO: 351) MTDLCPDAHCAKTHM VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 1284) (SEQ ID NO: 352) PYSRLCAYPCPDFVG LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 1285) (SEQ ID NO: 353) LCGCARSPDYCKCRG LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 1286) (SEQ ID NO: 354) WGRCERVPDCCCPRG LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 1287) (SEQ ID NO: 355) TRNTCHTRICYGMAC ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 1288) (SEQ ID NO: 356) CVCTSCSSYWTLCPD LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 1289) (SEQ ID NO: 357) LCCSRGSNCPDRCTW QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 1290) (SEQ ID NO: 358) CCPLCQANMCPDNQS LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 1291) (SEQ ID NO: 359) ECKLCCPDLYCGGTM QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 1292) (SEQ ID NO: 360) CSNPMCAYCCPDLIL ISSGLLSGRSGNH (SEQ ID NO: 1293) (SEQ ID NO: 361) CPRCNTYSKHDCYHQ ISSGLLSSGGSGGSLSGRNH (SEQ ID NO: 1294) (SEQ ID NO: 1091) FCCASKMPAPSNCHT ISSGLLSGRSANPRG (SEQ ID NO: 1295) (SEQ ID NO: 1092) AVGLLAPPTSGRSANPRG (SEQ ID NO: 1093) AVGLLAPPSGRSANPRG (SEQ ID NO: 1094) ISSGLLSGRSDDH (SEQ ID NO: 1095) ISSGLLSGRSDIH (SEQ ID NO: 1096) ISSGLLSGRSDQH (SEQ ID NO: 1097) ISSGLLSGRSDTH (SEQ ID NO: 1098) ISSGLLSGRSDYH (SEQ ID NO: 1099) ISSGLLSGRSDNP (SEQ ID NO: 1100) ISSGLLSGRSANP (SEQ ID NO: 1101) ISSGLLSGRSANI (SEQ ID NO: 1102) AVGLLAPPGGLSGRSDDH (SEQ ID NO: 1103) AVGLLAPPGGLSGRSDIH (SEQ ID NO: 1104) AVGLLAPPGGLSGRSDQH (SEQ ID NO: 1105) AVGLLAPPGGLSGRSDTH (SEQ ID NO: 1106) AVGLLAPPGGLSGRSDYH (SEQ ID NO: 1107) AVGLLAPPGGLSGRSDNP (SEQ ID NO: 1108) AVGLLAPPGGLSGRSANP (SEQ ID NO: 1109) AVGLLAPPGGLSGRSANI (SEQ ID NO: 1110) ISSGLLSGRSDNI (SEQ ID NO: 1111) AVGLLAPPGGLSGRSDNI (SEQ ID NO: 1112)

TABLE 13C VL VH VL CDRs VH CDRs SEQ SEQ SEQ ID NO: SEQ ID NO: Mask Sequence Substrates: ID NO: ID NO: CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 GCDFTSAKHNCGSGW LSGRSDNH 638 1514 VL CDRs of SEQ ID VH CDRs of SEQ ID (SEQ ID NO: 1351) (SEQ ID NO: 294) NO: 638 NO: 1514 VGSNCWTGPACALTS TGRGPSWV 1711 1712 1713 1714 1715 1716 (SEQ ID NO: 1352) (SEQ ID NO: 295) FCAVMFDFLSDRCLH PLTGRSGG (SEQ ID NO: 1353) (SEQ ID NO: 296) FCPPWLDYLGNKCMT TARGPSFK (SEQ ID NO: 1354) (SEQ ID NO: 297) MSCWDFSSAQGCGQH NTLSGRSENHSG (SEQ ID NO: 1355) (SEQ ID NO: 298) LMCADLHYNHYNCKY NTLSGRSGNHGS (SEQ ID NO: 1356) (SEQ ID NO: 299) ELCGWQSFSGVCTSE TSTSGRSANPRG (SEQ ID NO: 1357) (SEQ ID NO: 300) WTYENCWASCQPHLE TSGRSANP (SEQ ID NO: 1358) (SEQ ID NO: 301) KLTEDFSSAA (SEQ VHMPLGFLGP ID NO: 1359) (SEQ ID NO: 302) VGQSCFSGLVCDRQL AVGLLAPP (SEQ ID NO: 1360) (SEQ ID NO: 303) ISHYCFSGKSCRD AQNLLGMV (SEQ ID NO: 1361) (SEQ ID NO: 304) HCIPDFTSAAGDCMR QNQALRMA (SEQ ID NO: 1362) (SEQ ID NO: 305) RLVSAYSFS (SEQ ID LAAPLGLL NO: 1363) (SEQ ID NO: 306) KFHHSHPLVHDFTSA STFPFGMF (SEQ ID NO: 1364) (SEQ ID NO: 307) ASYPDFSSANGVGLR ISSGLLSS (SEQ ID NO: 1365) (SEQ ID NO: 308) GLATTLSNVDFTSAG PAGLWLDP (SEQ ID NO: 1366) (SEQ ID NO: 309) DFTSANSAFSGDAST VAGRSMRP (SEQ ID NO: 1367) (SEQ ID NO: 310) GRLPGHSVVDFTSAW VVPEGRRS (SEQ ID NO: 1368) (SEQ ID NO: 311) SGSFYSSSAFDFTSA ILPRSPAF (SEQ ID NO: 1369) (SEQ ID NO: 312) CDDFTSAQHSRINEC MVLGRSLL (SEQ ID NO: 1370) (SEQ ID NO: 313) CDFTSAQGKKCRTAL QGRAITFI (SEQ ID NO: 1371) (SEQ ID NO: 314) YYIDKYQSPSYGPVL SPRSIMLA (SEQ ID NO: 1372) (SEQ ID NO: 315) FSVARARSSADFTSS SMLRSMPL (SEQ ID NO: 1373) (SEQ ID NO: 316) DSDFTSAGSADSRSR SARGPSRW (SEQ ID NO: 1374) (SEQ ID NO: 319) CDFTSATSISKRCDH GWHTGRN (SEQ ID NO: 1375) (SEQ ID NO: 320) IESSASSWGLQASRN HTGRSGAL (SEQ ID NO: 1376) (SEQ ID NO: 321) PRYHNLNFTTPALSPGS AARGPAIH (SEQ ID NO: 1377) (SEQ ID NO: 322) DLFARFPLDRDFTSA RGPAFNPM (SEQ ID NO: 1378) (SEQ ID NO: 323) HCNFTTPPYCSSTLW SSRGPAYL (SEQ ID NO: 1379) (SEQ ID NO: 324) NVPIILLTDRQLLSG RGPATPIM (SEQ ID NO: 1380) (SEQ ID NO: 325) NPTACDFTSSMATYC RGPA (SEQ ID NO: 1381) (SEQ ID NO: 326) FVRTVRFSNSMFSVP GGQPSGMWGW (SEQ ID NO: 1382) (SEQ ID NO: 327) YDFSSASNSSPSRQT FPRPLGITGL (SEQ ID NO: 1383) (SEQ ID NO: 328) AHPDFSSAMRGNLLG SPLTGRSG (SEQ ID NO: 1384) (SEQ ID NO: 329) SSHVVHKDFTSANSR SAGFSLPA (SEQ ID NO: 1385) (SEQ ID NO: 330) CPDFTSANGGGCWQM LAPLGLQRR (SEQ ID NO: 1386) (SEQ ID NO: 331) SLGQSYPTDFTCPGC SGGPLGVR (SEQ ID NO: 1387) (SEQ ID NO: 332) ASMRSHEQRDFTSAY PLGL (SEQ ID NO: 1388) (SEQ ID NO: 333) SCQFWFTLCSGGVFH ISSGLSS (SEQ ID NO: 1389) (SEQ ID NO: 334) PYPNNRTGMHDFTSA PVGYTSSL (SEQ ID NO: 1390) (SEQ ID NO: 335) KPFPIDFTSAGTSGT DWLYWPGI (SEQ ID NO: 1391) (SEQ ID NO: 336) SIKSFIPRDDFTSAA LKAAPRWA (SEQ ID NO: 1392) (SEQ ID NO: 337) GIKNPATPFVDFTSA GPSHLVLT (SEQ ID NO: 1393) (SEQ ID NO: 338) LSHTYPRGSSTIEAS LPGGLSPW (SEQ ID NO: 1394) (SEQ ID NO: 339) PSLDFSSAT (SEQ ID MGLFSEAG NO: 1395) (SEQ ID NO: 340) AFTPRIAPTFDVMKE SPLPLRVP (SEQ ID NO: 1396) (SEQ ID NO: 341) LCGLQIPPDCERS RMHLRSLG (SEQ ID NO: 1397) (SEQ ID NO: 342) AAKMVSHSERDFTSA LLAPSHRA (SEQ ID NO: 1398) (SEQ ID NO: 343) VSVECFSGMQCPHYY GPRSFGL (SEQ ID NO: 1399) (SEQ ID NO: 344) ASKCRLPCMASTQIY GPRSFG (SEQ ID NO: 1400) (SEQ ID NO: 345) GLRSCNIYFSIPCTY LSGRSGNH (SEQ ID NO: 1401) (SEQ ID NO: 1157) RGTSDGTLDFTTARS SGRSANPRG (SEQ ID NO: 1402) (SEQ ID NO: 1158) SMYPSASRLLHPQYP LSGRSDDH (SEQ ID NO: 1403) (SEQ ID NO: 1161) HCISCYDFTSAAGSF LSGRSDIH (SEQ ID NO: 1404) (SEQ ID NO: 1162) SSGRWGDAWACARIC LSGRSDQH (SEQ ID NO: 1405) (SEQ ID NO: 1165) RVFSDFTSASHSFGG LSGRSDTH (SEQ ID NO: 1406) (SEQ ID NO: 1166) TDRHSASGRDFTSAH LSGRSDYH (SEQ ID NO: 1407) (SEQ ID NO: 1169) AHCEDFSSAERIATMGC LSGRSDNP (SEQ ID NO: 1408) (SEQ ID NO: 1520) ACDPYSFSIPCDDRL LSGRSANP (SEQ ID NO: 1409) (SEQ ID NO: 1695) NSPFTLSHIYDR (SEQ LSGRSANI ID NO: 1410) (SEQ ID NO: 1696) IGINFTTPSAFVAFP LSGRSDNI (SEQ ID NO: 1411) (SEQ ID NO: 1697) RDAFPIYRNADFSTP MIAPVAYR (SEQ ID NO: 1412) (SEQ ID NO: 1698) SIPNASSYNFTSSSG RPSPMWAY (SEQ ID NO: 1413) (SEQ ID NO: 1699) AGIPDKRHTYDFTSA WATPRPMR (SEQ ID NO: 1414) (SEQ ID NO: 1700) WPLAHDSRDWNFTTP FRLLDWQW (SEQ ID NO: 1415) (SEQ ID NO: 1701) RHSPSSGHVDFTSAG ISSGL (SEQ ID NO: 1416) (SEQ ID NO: 1702) SCFAWTDPVWNRCSW ISSGLLS (SEQ ID NO: 1417) (SEQ ID NO: 1703) MPCDWTGPGKIWCGG ISSGLL (SEQ ID NO: 1418) (SEQ ID NO: 1704) RDCDFSTANFRSCNK ISSGLLSGRSDNH (SEQ ID NO: 1419) (SEQ ID NO: 214) LSCVVSPNYLHCNDH AVGLLAPPGGLSGRSDNH (SEQ ID NO: 1420) (SEQ ID NO: 318) FVCGLYSFSGVCQGV ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 1421) (SEQ ID NO: 346) IGLMCFSGLQCPMLA ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 1422) (SEQ ID NO: 346) PGMNCFSGEICQMST AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 1423) (SEQ ID NO: 347) GDVGSCWASCGLQGG TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 1424) (SEQ ID NO: 348) SQFQDCWASCGASFT VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 1425) (SEQ ID NO: 349) VGSLNCWYSCGDIWL TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 1426) (SEQ ID NO: 350) MCESWLNFLGDQCGM LSGRSDNHGGAVGLLAPP (SEQ ID NO: 1427) (SEQ ID NO: 351) RCMISQSSFSGMCGM VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 1428) (SEQ ID NO: 352) NCAPWTSNMSNHCLK LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 1429) (SEQ ID NO: 353) LCGVGSATGLELCGV LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 1430) (SEQ ID NO: 354) GCDFSSLGGRQPCTP LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 1431) (SEQ ID NO: 355) MGCNFTTYPYHTCNT ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 1432) (SEQ ID NO: 356) GSCDFTSGAGKKCGS LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 1433) (SEQ ID NO: 357) VSCDFTSSHARMCSR QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 1434) (SEQ ID NO: 358) MRCIDFYYNHINCIG LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 1435) (SEQ ID NO: 359) RSCDFTSAANKYCAT QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 1436) (SEQ ID NO: 360) LYCDSFSVPRPNCAP ISSGLLSGRSGNH (SEQ ID NO: 1437) (SEQ ID NO: 361) NSCDFTSARVSKCST ISSGLLSSGGSGGSLSGRNH (SEQ ID NO: 1438) (SEQ ID NO: 1091) STCSDNFTTPMPCNT ISSGLLSGRSANPRG (SEQ ID NO: 1439) (SEQ ID NO: 1092) DICNDRPNLTHCHYF AVGLLAPPTSGRSANPRG (SEQ ID NO: 1440) (SEQ ID NO: 1093) LRCDDFTSAIGCRGY AVGLLAPPSGRSANPRG (SEQ ID NO: 1441) (SEQ ID NO: 1094) EGCDFTSALHSCNNY ISSGLLSGRSDDH (SEQ ID NO: 1442) (SEQ ID NO: 1095) RKGCGDFTSASCFVV ISSGLLSGRSDIH (SEQ ID NO: 1443) (SEQ ID NO: 1096) GMLCAGSSFGLCESM ISSGLLSGRSDQH (SEQ ID NO: 1444) (SEQ ID NO: 1097) RESCFGSSLGLCTNK ISSGLLSGRSDTH (SEQ ID NO: 1445) (SEQ ID NO: 1098) ILRCYDIPTNCMNFN ISSGLLSGRSDYH (SEQ ID NO: 1446) (SEQ ID NO: 1099) NSECTFGAMYCRNKP ISSGLLSGRSDNP (SEQ ID NO: 1447) (SEQ ID NO: 1100) ASGCFDEDIRCSGGA ISSGLLSGRSANP (SEQ ID NO: 1448) (SEQ ID NO: 1101) HYFCNQSNPSCQTAP ISSGLLSGRSANI (SEQ ID NO: 1449) (SEQ ID NO: 1102) AMGCELGGAGCIGSP AVGLLAPPGGLSGRSDDH (SEQ ID NO: 1450) (SEQ ID NO: 1103) TLKCHMPRKLCANDP AVGLLAPPGGLSGRSDIH (SEQ ID NO: 1451) (SEQ ID NO: 1104) RPACRDLPHNCITST AVGLLAPPGGLSGRSDQH (SEQ ID NO: 1452) (SEQ ID NO: 1105) QMSCHGNFTTCHSNP AVGLLAPPGGLSGRSDTH (SEQ ID NO: 1453) (SEQ ID NO: 1106) LTGCARGARPCRLRV AVGLLAPPGGLSGRSDYH (SEQ ID NO: 1454) (SEQ ID NO: 1107) WSELCLAGPSCGWVG AVGLLAPPGGLSGRSDNP (SEQ ID NO: 1455) (SEQ ID NO: 1108) VTKSCWQLPHCITAP AVGLLAPPGGLSGRSANP (SEQ ID NO: 1456) (SEQ ID NO: 1109) KAASCPHNQICNMTA AVGLLAPPGGLSGRSANI (SEQ ID NO: 1457) (SEQ ID NO: 1110) VSKNCFSGMMCPVFA ISSGLLSGRSDNI (SEQ ID NO: 1458) (SEQ ID NO: 1111) NRSSCWTGPTCHVLH AVGLLAPPGGLSGRSDNI (SEQ ID NO: 1459) (SEQ ID NO: 1112) ARTGCSGPVCLNDVS (SEQ ID NO: 1460) STRTCLAFTCINGNT (SEQ ID NO: 1461) MLDGNCWYACSYKNT (SEQ ID NO: 1462) FSRSDCWSACAPWRV (SEQ ID NO: 1463) GGRMDCWASCQPLSR (SEQ ID NO: 1464) NSPHSCMTNCDFTSA (SEQ ID NO: 1465)

TABLE 13D LC CDRs HC CDRs LC HC SEQ ID NO: SEQ ID NO: Mask Sequence Substrates: SEQ ID NO: SEQ ID NO: CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 DYTYCRWVNWCLSGV LSGRSDNH 543 546 LC CDRs of HC CDRs of (SEQ ID NO: 384) (SEQ ID NO: 294) SEQ ID NO: 543 SEQ ID NO: 546 ILCPEDPWGHKCKLP TGRGPSWV (SEQ ID NO: 385) (SEQ ID NO: 295) TNIWSCQTYCDHKHK PLTGRSGG (SEQ ID NO: 386) (SEQ ID NO: 296) SDHKCKLQNCMNTKV TARGPSFK (SEQ ID NO: 387) (SEQ ID NO: 297) PGNCHPMQKEMCQFI NTLSGRSENHSG (SEQ ID NO: 388) (SEQ ID NO: 298) VEHLCYTHNKCKHPD NTLSGRSGNHGS (SEQ ID NO: 389) (SEQ ID NO: 299) TIPRCGQHPKCKDTL TSTSGRSANPRG (SEQ ID NO: 390) (SEQ ID NO: 300) ACRICQDHPKTKWNS TSGRSANP (SEQ ID NO: 391) (SEQ ID NO: 301) LIQCTGNLDHKCKHY VHMPLGFLGP (SEQ ID NO: 392) (SEQ ID NO: 302) IPCHHSADHKHKCTS AVGLLAPP (SEQ ID NO: 393) (SEQ ID NO: 303) SRQICADYNCHNKYK AQNLLGMV (SEQ ID NO: 394) (SEQ ID NO: 304) QPCNPQIDHKIKCIY QNQALRMA (SEQ ID NO: 395) (SEQ ID NO: 305) HYTICMTHNKCKDMA LAAPLGLL (SEQ ID NO: 396) (SEQ ID NO: 306) ANSCLAVEHKCKHNY STFPFGMF (SEQ ID NO: 397) (SEQ ID NO: 307) AALHCTEHKCKNHIK ISSGLLSS (SEQ ID NO: 398) (SEQ ID NO: 308) APCIINTVDWKSCEI PAGLWLDP (SEQ ID NO: 399) (SEQ ID NO: 309) ATNWCTHKQKCKQDM VAGRSMRP (SEQ ID NO: 400) (SEQ ID NO: 310) DCYNEHKLKTRVCNN VVPEGRRS (SEQ ID NO: 401) (SEQ ID NO: 311) DEMQCSHKQKCTNSK ILPRSPAF (SEQ ID NO: 402) (SEQ ID NO: 312) DVGICSQHNKCKPTK MVLGRSLL (SEQ ID NO: 403) (SEQ ID NO: 313) EKYCSSDDHKCKITL QGRAITFI (SEQ ID NO: 404) (SEQ ID NO: 314) ELECSHNKVKNCIQI SPRSIMLA (SEQ ID NO: 405) (SEQ ID NO: 315) ELHPCNTHKCKPIVN SMLRSMPL (SEQ ID NO: 406) (SEQ ID NO: 316) EVGSCNHPKCKSNNY SARGPSRW (SEQ ID NO: 407) (SEQ ID NO: 319) EYSPSLAHPKLKDNA GWHTGRN (SEQ ID NO: 408) (SEQ ID NO: 320) FESLHPKGKHPEDLG HTGRSGAL (SEQ ID NO: 409) (SEQ ID NO: 321) FPLCVRADRVCGDAQ AARGPAIH (SEQ ID NO: 410) (SEQ ID NO: 322) FQAPPASHNKLKPSL RGPAFNPM (SEQ ID NO: 411) (SEQ ID NO: 323) GAIDSCHHKCKSPHY SSRGPAYL (SEQ ID NO: 412) (SEQ ID NO: 324) GKIYTCEHNCTFGYS RGPATPIM (SEQ ID NO: 413) (SEQ ID NO: 325) HCTVNNHSSDHKCKI RGPA (SEQ ID NO: 414) (SEQ ID NO: 326) HGTQCTHNKCKPILS GGQPSGMWGW (SEQ ID NO: 415) (SEQ ID NO: 327) HIGWCLHPKCKTTTT FPRPLGITGL (SEQ ID NO: 416) (SEQ ID NO: 328) HLRTCIQKWCEHKMK SPLTGRSG (SEQ ID NO: 417) (SEQ ID NO: 329) HTDCTMMSNHKCKIN SAGFSLPA (SEQ ID NO: 418) (SEQ ID NO: 330) IRQQCTALACLLKVH LAPLGLQRR (SEQ ID NO: 419) (SEQ ID NO: 331) KGCSTHKMRAYCNQM SGGPLGVR (SEQ ID NO: 420) (SEQ ID NO: 332) KMFTPCKIWCNNSYN PLGL (SEQ ID NO: 421) (SEQ ID NO: 333) KTMCSGHKQKCNNSS ISSGLSS (SEQ ID NO: 422) (SEQ ID NO: 334) LACHSASLVDHKCKL PVGYTSSL (SEQ ID NO: 423) (SEQ ID NO: 335) LCNVSMDHKHKPCYL DWLYWPGI (SEQ ID NO: 424) (SEQ ID NO: 336) LGLNCFSEHKCKEHM LKAAPRWA (SEQ ID NO: 425) (SEQ ID NO: 337) LGTCTHKHKNCNYTL GPSHLVLT (SEQ ID NO: 426) (SEQ ID NO: 338) LHEGCTTHNKCKPIA LPGGLSPW (SEQ ID NO: 427) (SEQ ID NO: 339) LKRSCTGHWTCYTNW MGLFSEAG (SEQ ID NO: 428) (SEQ ID NO: 340) LQRCTHKEKYCHAIH SPLPLRVP (SEQ ID NO: 429) (SEQ ID NO: 341) LSHCYDHKRKCSYIV RMHLRSLG (SEQ ID NO: 430) (SEQ ID NO: 342) LSKCHNKEKNCSNNN LLAPSHRA (SEQ ID NO: 431) (SEQ ID NO: 343) MDTCEMHKQKCRPSF GPRSFGL (SEQ ID NO: 432) (SEQ ID NO: 344) MHNECLTHKCKVPIT GPRSFG (SEQ ID NO: 433) (SEQ ID NO: 345) MLTLCNTNACHKEKN LSGRSGNH (SEQ ID NO: 434) (SEQ ID NO: 1157) MRPCLNNLEHKCKHY SGRSANPRG (SEQ ID NO: 435) (SEQ ID NO: 1158) MSRCPTHKMKCSLNI LSGRSDDH (SEQ ID NO: 436) (SEQ ID NO: 1161) MWICQEHKLKCMTDT LSGRSDIH (SEQ ID NO: 437) (SEQ ID NO: 1162) MYYCKRRSAFYCTLN LSGRSDQH (SEQ ID NO: 438) (SEQ ID NO: 1165) NDCQHDKQMHKCKMH LSGRSDTH (SEQ ID NO: 439) (SEQ ID NO: 1166) NFGPCPMLLGCFGFR LSGRSDYH (SEQ ID NO: 440) (SEQ ID NO: 1169) NHTDCSHPKCKSHDS LSGRSDNP (SEQ ID NO: 441) (SEQ ID NO: 1520) NLNCPHKQKNCDKYH LSGRSANP (SEQ ID NO: 442) (SEQ ID NO: 1695) NPQCTPIDHKCKTHH LSGRSANI (SEQ ID NO: 443) (SEQ ID NO: 1696) NTTSCTHPKCKHQGK LSGRSDNI (SEQ ID NO: 444) (SEQ ID NO: 1697) NVGGCDNYGCHKLKN MIAPVAYR (SEQ ID NO: 445) (SEQ ID NO: 1698) PCSPGNLTWDHKCKY RPSPMWAY (SEQ ID NO: 446) (SEQ ID NO: 1699) PFTKCHGFNKCKEHT WATPRPMR (SEQ ID NO: 447) (SEQ ID NO: 1700) PGDKCTHKEKCYYNN FRLLDWQW (SEQ ID NO: 448) (SEQ ID NO: 1701) PNICNLDHKRKCRIN ISSGL (SEQ ID NO: 449) (SEQ ID NO: 1702) PQLACKHPKCKDAGN ISSGLLS (SEQ ID NO: 450) (SEQ ID NO: 1703) PSCTMWTHGGVCKHA ISSGLL (SEQ ID NO: 451) (SEQ ID NO: 1704) PSHRHPLAKPGFRVE ISSGLLSGRSDNH (SEQ ID NO: 452) (SEQ ID NO: 214) PTCFKTHNKSKCNRV AVGLLAPPGGLSGRSDNH (SEQ ID NO: 453) (SEQ ID NO: 318) PTPVCHHNFHCFGYD ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 454) (SEQ ID NO: 346) QATCQWKKRSKCHNK ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 455) (SEQ ID NO: 346) QHSWCQHKAKCNYGN AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 456) (SEQ ID NO: 347) QNCSPTYTTHKCKLT TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 457) (SEQ ID NO: 348) QSSNCEHKRKCSSIS VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 458) (SEQ ID NO: 349) RPCLLGLVPDHKCKL TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 459) (SEQ ID NO: 350) RRSCMRSINTCKQKY LSGRSDNHGGAVGLLAPP (SEQ ID NO: 460) (SEQ ID NO: 351) RSSCPTVTPQNCENQ VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 461) (SEQ ID NO: 352) RTMCLDLNHKCKPSN LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 462) (SEQ ID NO: 353) RTYWCTNHNKCKHNM LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 463) (SEQ ID NO: 354) RVENCEHNQYCHKWK LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 464) (SEQ ID NO: 355) SCHEDDHKNKNICSL ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 465) (SEQ ID NO: 356) SDTCVMNHPKCKRDN LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 466) (SEQ ID NO: 357) SSTCFHPNQKECMTK QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 467) (SEQ ID NO: 358) SSYCGGITMRCRRAM LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 468) (SEQ ID NO: 359) STGYCTYVNWCNYTN QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 469) (SEQ ID NO: 360) THKCKLHLQVCTQTT ISSGLLSGRSGNH (SEQ ID NO: 470) (SEQ ID NO: 361) TMNCTHPKQKCQHTN ISSGLLSSGGSGGSLSGRNH (SEQ ID NO: 471) (SEQ ID NO: 1091) TNVLCESHNCDHKNK ISSGLLSGRSANPRG (SEQ ID NO: 472) (SEQ ID NO: 1092) TQHAASLGVEHKSKI AVGLLAPPTSGRSANPRG (SEQ ID NO: 473) (SEQ ID NO: 1093) TQLPCFDDHKCKNTN AVGLLAPPSGRSANPRG (SEQ ID NO: 474) (SEQ ID NO: 1094) TSDSCMRQKCEHKEK ISSGLLSGRSDDH (SEQ ID NO: 475) (SEQ ID NO: 1095) TTCDDHKYKHKCAQL ISSGLLSGRSDIH (SEQ ID NO: 476) (SEQ ID NO: 1096) TTCGAHKEKQHCIYT ISSGLLSGRSDQH (SEQ ID NO: 477) (SEQ ID NO: 1097) TTYCAYWHNKCKFET ISSGLLSGRSDTH (SEQ ID NO: 478) (SEQ ID NO: 1098) VGPTCGHAKCKQSEV ISSGLLSGRSDYH (SEQ ID NO: 479) (SEQ ID NO: 1099) VSHPCNTHKCKTNIV ISSGLLSGRSDNP (SEQ ID NO: 480) (SEQ ID NO: 1100) WDCRNTSHPKLKCHN ISSGLLSGRSANP (SEQ ID NO: 481) (SEQ ID NO: 1101) WSPCNSDHKRKCNNG ISSGLLSGRSANI (SEQ ID NO: 482) (SEQ ID NO: 1102) YANMSCEYDCHKMKY AVGLLAPPGGLSGRSDDH (SEQ ID NO: 483) (SEQ ID NO: 1103) YANPCTHKEKCHFKN AVGLLAPPGGLSGRSDIH (SEQ ID NO: 484) (SEQ ID NO: 1104) YDCSPSWTHPKCKHK AVGLLAPPGGLSGRSDQH (SEQ ID NO: 485) (SEQ ID NO: 1105) YGWTCTTHPKCKTTN AVGLLAPPGGLSGRSDTH (SEQ ID NO: 486) (SEQ ID NO: 1106) YQKCHPKAKDCGNNT AVGLLAPPGGLSGRSDYH (SEQ ID NO: 487) (SEQ ID NO: 1107) YWECPNMEHNKCKNN AVGLLAPPGGLSGRSDNP (SEQ ID NO: 488) (SEQ ID NO: 1108) PMGNRYCVLDHPKLK AVGLLAPPGGLSGRSANP (SEQ ID NO: 489) (SEQ ID NO: 1109) GHKSCCQKHCEYTQT AVGLLAPPGGLSGRSANI (SEQ ID NO: 490) (SEQ ID NO: 1110) LYLEMCSCCCWESIT ISSGLLSGRSDNI (SEQ ID NO: 491) (SEQ ID NO: 1111) ACQAQHCYKTYACKP AVGLLAPPGGLSGRSDNI (SEQ ID NO: 492) (SEQ ID NO: 1112) CCYTCSVHPKCKNQL (SEQ ID NO: 493) CKHRCSHKEKCPANH (SEQ ID NO: 494) CHVLFCLMQCCRWSL (SEQ ID NO: 495) LNSSLVFDHPKAKPN (SEQ ID NO: 496) MCLLCRSKFGCKVKG (SEQ ID NO: 497) IICNDHKCKQNQCNN (SEQ ID NO: 498) IRCSLRDSLCGCERM (SEQ ID NO: 499) TSCQPPKHKCTCNHG (SEQ ID NO: 500) TQCPHRCVKPNCWLH (SEQ ID NO: 501) KCCETKRNHKHCTYK (SEQ ID NO: 502) LPHCCHKAKHCNHTS (SEQ ID NO: 503) PAMCAAIHEKCCIKV (SEQ ID NO: 504) PRSCGNQLCPCHYYK (SEQ ID NO: 505) TNKCSCNHNMKCINY (SEQ ID NO: 506) VETCCQHNKCKYPFI (SEQ ID NO: 507) IFCCSNHEDHKCKTN (SEQ ID NO: 508) VCRLICPLTCVIGVG (SEQ ID NO: 509) FHGCCSVYSCLTNPG (SEQ ID NO: 510) ALACHPKQKPLEGQL (SEQ ID NO: 511) SIICCATSSCPLKHL (SEQ ID NO: 512) APCCRPHKEKPIDSR (SEQ ID NO: 513) WELCCPSADCRVAMG (SEQ ID NO: 514) QDHPKTKWNS (SEQ ID NO: 548) ACRICQDHPATKWNS (SEQ ID NO: 549) ACRICQDHPKTAWNS (SEQ ID NO: 550) ACRICQDAPKTKWNS (SEQ ID NO: 551) ACRICQDHAKTKWNS (SEQ ID NO: 552) DHPATKWNS (SEQ ID NO: 553) DHPKTAWNS (SEQ ID NO: 554) DAPKTKWNS (SEQ ID NO: 555) DAPATKWNS (SEQ ID NO: 556) ACRICQDHP (SEQ ID NO: 557) HPQSKDTL (SEQ ID NO: 558) HPKSQDTL (SEQ ID NO: 559) TIPRCGQHPLCLDTL (SEQ ID NO: 560) HPLSLDTL (SEQ ID NO: 561) HPASKDTL (SEQ ID NO: 562) HPKSADTL (SEQ ID NO: 563) PGNCHPLQKELCQFI (SEQ ID NO: 564) HPLQKELAQFI (SEQ ID NO: 565) HPLALELAQFI (SEQ ID NO: 566) PGNCHPLQLELCQFI (SEQ ID NO: 567) TNIWSCQTYCDHAHA (SEQ ID NO: 568) TNIWSCQTYCDHAHL (SEQ ID NO: 569) TNIWSCQTYCDHLHA (SEQ ID NO: 570) TNIWSCQTYCDHKHA (SEQ ID NO: 571)

Any of the combinations described in Tables 13A, 13B, 13C, and/or 13D can be combined with human immunoglobulin constant regions to result in fully humanized IgGs including IgG1, IgG2, IgG4 or mutated constant regions to result in human IgGs with altered functions such as IgG1 N297A, IgG1 N297Q, or IgG4 S228P. Additional examples are known to those skilled in the art. Examples of Ig heavy chain constant region amino acids in which mutations in at least one amino acid leads to reduced Fc function include, but are not limited to, mutations in amino acid 228, 233, 234, 235, 236, 237, 239, 252, 254, 256, 265, 270, 297, 318, 320, 322, 327, 329, 330, and 331 of the heavy constant region. Examples of combinations of mutated amino acids are also known in the art, such as, but not limited to a combination of mutations in amino acids 234, 235, and 331, such as L234F, L235E, and P331S or a combination of amino acids 318, 320, and 322, such as E318A, K320A, and K322A.

As an example a masking moiety comprising SEQ ID NO: 66 can be combined with a substrate comprising SEQ ID NO: 294, and a VL region comprising SEQ ID NO: 39 in combination with a human kappa constant domain comprising SEQ ID NO: 61 to produce a light chain, which may or may not include linker regions. One such example is SEQ ID NO: 380, although other embodiments having longer linker sequences are also contemplated. In some embodiments, for example, a VH region comprising SEQ ID NO: 21 can be combined a with human immunoglobulin heavy chain constant domain to give an IgG1 heavy chain (SEQ ID NO: 2048), an IgG4 heavy chain (SEQ ID NO: 2051), an IgG4 S228P heavy chain (SEQ ID NO: 2047), a mutated IgG1 N297A heavy chain (SEQ ID NO: 2049) or a mutated IgG1 N297Q heavy chain (SEQ ID NO: 2050). Co-expression of SEQ ID NO: 380 with SEQ ID NO: 2048 will yield an IgG1 anti-PD-1 activatable antibody. Co-expression of SEQ ID NO: 380 with SEQ ID NO: 2051 will yield an IgG4 anti-PD-1 activatable antibody. Co-expression of SEQ ID NO: 380 with SEQ ID NO: 2047 will yield an IgG4S228P anti-PD-1 activatable antibody. Co-expression of SEQ ID NO: 380 with SEQ ID NO: 2049 will yield an IgG1 N297A anti-PD-1 activatable antibody. Co-expression of SEQ ID NO: 380 with SEQ ID NO: 2050 will yield an IgG1 N297Q anti-PD-1 activatable antibody.

In some embodiments, a masking moiety comprising SEQ ID NO: 99 is combined with a substrate comprising SEQ ID NO: 214 and a VL region comprising SEQ ID NO: 47 in combination with a human kappa constant domain comprising SEQ ID NO: 61 to produce a light chain comprising SEQ ID NO: 2055. In some embodiments, spacer QGQSGQG (SEQ ID NO: 362) is added to the N-terminus of SEQ ID NO: 2055 to form SEQ ID NO: 2054. In some embodiments, a masking moiety comprising SEQ ID NO: 99 is combined with a substrate comprising SEQ ID NO: 1100 and a VL region comprising SEQ ID NO: 47 in combination with a human kappa constant domain comprising SEQ ID NO: 61 to produce a light chain comprising SEQ ID NO: 2057. In some embodiments, spacer QGQSGQG (SEQ ID NO: 362) is added to the N-terminus of SEQ ID NO: 2057 to form SEQ ID NO: 2056. In some embodiments, a masking moiety comprising SEQ ID NO: 99 is combined with a substrate comprising SEQ ID NO: 1101, and a VL region comprising SEQ ID NO: 47 in combination with a human kappa constant domain comprising SEQ ID NO: 61 to produce a light chain comprising SEQ ID NO: 2059. In some embodiments, spacer QGQSGQG (SEQ ID NO: 362) is added to the N-terminus of SEQ ID NO: 2059 to form SEQ ID NO: 2058. In some embodiments, a VH region comprising SEQ ID NO: 21 can be combined with an IgG4 S228P human immunoglobulin heavy chain constant domain comprising SEQ ID NO: 63 to produce a heavy chain comprising SEQ ID NO: 2052. In some embodiments, the C-terminal lysine of SEQ ID NO: 2052 is missing to form an amino acid sequence having SEQ ID NO: 2053. Co-expression of any one of these light chains with any one of these heavy chains will lead to production of an activatable antibody of the embodiments.

Constant region amino acid sequences are shown below in SEQ ID NO: 381, 382, 383, and 1807.

IgG1 Hc CH1-end amino acid sequence: (SEQ ID NO: 381) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

STYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG IgG1NA CH1-end amino acid sequence: (SEQ ID NO: 382) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

STYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG IgG1NQ CH1-end amino acid sequence: (SEQ ID NO: 1807) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

STYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG IgG4 Hc CH1-end amino acid sequence: (SEQ ID NO: 383) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLG Light Chain (Lc) amino acid sequence: AMSGCSWSAFCPYLA[X1]_(n)LSGRSDNH[X2]_(n)DIQLTQSPSSLSASVGDRVTITCRASESVD NYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYC QQSKDVPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC where each of [X1]_(n) and [X2]_(n) independently can be a linking peptide of between 0 and 20 amino acids (SEQ ID NO: 380), or any other suitable length. IgG4 S228P Heavy Chain (Hc) amino acid sequence: (SEQ ID NO: 2047) EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVAYISNSGGNAHYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREDYGTSPFVYWGQGTLVTVSSASTKGPSVF PLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgG1 Heavy Chain (Hc) amino acid sequence: (SEQ ID NO: 2048) EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVAYISNSGGNAHYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREDYGTSPFVYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

STYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG IgG1NA Hc amino acid sequence: (SEQ ID NO: 2049) EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVAYISNSGGNAHYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREDYGTSPFVYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

STYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG IgG1NQ Hc amino acid sequence: (SEQ ID NO: 2050) EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVAYISNSGGNAHYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREDYGTSPFVYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

STYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG IgG4 Hc amino acid sequence: (SEQ ID NO: 2051) EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVAYISNSGGNAHYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREDYGTSPFVYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTIPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLG Heavy Chain 1 Amino Acid Sequence (SEQ ID NO: 2052): EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVAYISNSGGNA HYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREDYGTSPFVYWGQGTLVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Heavy Chain 2 Amino Acid Sequence (SEQ ID NO: 2053): EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVAYISNSGGNA HYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTREDYGTSPFVYWGQGTLVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFScSVMHEALHNHYTQKSLSLSLG Light Chain 1 Amino Acid Sequence with spacer (SEQ ID NO: 2054): [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSSLS ASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGS GTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC] Light Chain 1 Amino Acid Sequence without spacer (SEQ ID NO: 2055): TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSSLSASVGDRVTI TCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISS MQPEDFATYYCQQSKDVPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC Light Chain 2 Amino Acid Sequence with spacer (SEQ ID NO: 2056): [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDNPGGGSDIQLTQSPSSLS ASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGS GTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC] Light Chain 2 Amino Acid Sequence without spacer (SEQ ID NO: 2057): TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDNPGGGSDIQLTQSPSSLSASVGDRVTIT CRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSM QPEDFATYYCQQSKDVPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC Light Chain 3 Amino Acid Sequence with spacer (SEQ ID NO: 2058): [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSANPGGGSDIQLTQSPSSLS ASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGS GTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC] Light Chain 3 Amino Acid Sequence without spacer (SEQ ID NO: 2059): TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSANPGGGSDIQLTQSPSSLSASVGDRVTIT CRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSM QPEDFATYYCQQSKDVPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

Example 10: Evaluation of Efficiency of Masking Moieties

This example describes activatable anti-PD-1 antibodies of the disclosure with reduced binding to hPD-1.

Masking the ability of an antibody to bind to its antigen is an example of inhibition of binding. The extent of inhibition is dependent on the affinity of the antibody for its antigen, the affinity of the masking moiety (also referred to herein as the mask) for the antibody and the concentration of all reactants. Local concentrations of the tethered peptide mask (inhibitor) is very high in the activatable antibody context, on the order of 10 mM, therefore moderate affinity peptides would effectively mask activatable antibody antigen binding.

Masking efficiencies were evaluated by standard plate ELISA. Briefly, human PD-1-Fc (R and D systems, Minneapolis, Minn.) was adsorbed to the wells of a 96-well ELISA plate. Purified anti-PD-1 antibodies and activatable antibodies were applied to the plate in serial dilution and allowed to bind. Bound antibody and activatable antibodies were detected with anti-human IgG-HRP conjugate, FAb-specific (Sigma, St Louis, Mo.) and visualized with the chromogenic substrate TMB (Thermo Scientific, Rockford, Ill.). Shown are plots of binding isotherms for anti-PD-1 A1.4 and anti-PD-1 A1.5 activatable antibodies (FIGS. 14-18). Plots were generated in Prizm (Sigma Plot); the data were fit to a model of single site saturation binding, and the equilibrium dissociation constant, K_(d), was determined. Masking efficiency was calculated by dividing the K_(d) for binding of the activatable antibodies by the K_(d) of the parental antibody. The masking efficiency (ME) values for the tested antibodies are shown in Table 14 and Table 15 below (masking moiety PD001 is also referred to herein as PD01 and/or PD-01; masking moiety PD002 is also referred to herein as PD02 and/or PD-02, and so on), and the apparent K_(d) (nM) and masking efficiency (ME) values for the tested activatable antibodies are shown in Table 26:

TABLE 14 Masking efficiency values for activatable antibodies tested in FIG. 14 Mask ME PD01 25 PD02 18 PD03  5 PD08 13 PD09  2  PD-10 13

TABLE 15 Masking efficiency values for activatable antibodies tested in FIG. 18 Mask ME None  1 PD06 30 PD09  2  PD-12 10  PD-17 20  PD-19 30 PD25 30 PD26 20 PD28 25 PD34 42

TABLE 26 Apparent Kd and Masking efficiency values for activatable antibodies tested in FIG. 15 Molecule Apparent KD, nM Masking Efficiency A1.5 (n = 5 avg) 1.8 PD01 348 193 PD02 21 12 PD03 109 61 PD05 68 38 PD06 71 39 PD08 55 31 PD09 10 6 PD10 54 30 PD11 67 37 PD12 25 14 PD13 151 84 PD14 100 56 PD15 117 65 PD16 57 32 PD17 22 12 PD18 75 42 PD19 93 52 PD20 77 43 PD21 49 27 PD22 41 23 PD23 31 17 PD24 92 51

Example 11: Anti-PD-1 Activatable Antibodies of the Embodiments are Functionally Masked in a Human T-Cell Restimulation Assay

This example describes the effect of masking moieties on the biological function of the anti-PD-1 antibody. (FIG. 19)

PBMCs from a CMV-positive donor (Hemacare) were plated at 2×10⁵ cells/well in the presence of CMV viral lysate (Astarte) and either anti-PD-1 antibody A1.5, an anti-PD-1 activatable antibody of the disclosure, or hIgG4 isotype control antibody. After four days, supernatant was removed from each well and IFN-gamma levels were assayed using IFN-gamma ELISA kit (Life Technologies, Carlsbad, Calif.). Anti-PD-1 A1.5 activatable antibodies increased CMV-stimulated IFN-gamma secretion compared with control hIgG4 but with decreased potency relative to anti-PD-1 A1.5 parental antibody (FIG. 19).

Example 12: Anti Mouse PD-1 J43v2 mIgG2a Antibody Binds Mouse PD-1 and Blocks Mouse PD-L1 and Mouse PD-L2 Binding

This example demonstrates that Armenian hamster anti mouse PD-1 J43 antibody (U.S. Pat. No. 7,858,746; Agata et al, 1996, International Immunology, Vol. 8 No. 5 pp 765-77) can be functionally expressed as a mouse IgG2a antibody.

Anti-mouse PD-1 antibody J43 was reformatted as a hamster LC (SEQ ID NO: 543 amino acid; SEQ ID NO: 544 or SEQ ID NO: 545 nucleotide), mouse IgG2a antibody by fusion of the heavy chain variable domain to mIgG2a, resulting in amino acid SEQ ID NO 546, nucleotide sequence SEQ ID NO 547. Full length J43v2 mIgG2a (also referred to as J43v2, J43 m2a and J43v2 m2a) was expressed by transient transfection of HEK293 cells and purified from the culture supernatant by Protein G chromatography. One embodiment of this disclosure is an antibody comprising a light chain comprising amino acid sequence SEQ ID NO: 543. One embodiment of this disclosure is an antibody comprising a heavy chain comprising amino acid sequence SEQ ID NO: 546. One embodiment of this disclosure is an antibody comprising a light chain comprising amino acid sequence SEQ ID NO: 543 and a heavy chain comprising amino acid sequence SEQ ID NO: 546. One embodiment of this disclosure is an antibody comprising the CDRs of an antibody comprising a light chain comprising amino acid sequence SEQ ID NO: 543 and a heavy chain comprising amino acid sequence SEQ ID NO: 546.

Functionality was assayed by binding of J43v2 m2a to HEK293 cells expressing mouse PD-1 and confirmation that binding of J43v2 m2a to HEK293-mPD-1 inhibited binding of biotinylated mPD-L1 and biotinylated mPD-L2 to HEK293-mPD-1 (FIG. 20). About 100,000 mPD-1 HEK293 cells were transferred to a U-bottom 96-well plate. For the binding experiment, a 1:4 titration of J43v2 m2a antibody starting at 150 nM was added to cells. For blocking experiments, 20 nM of biotinylated-mPD-L1-Fc or 20 nM of biotinylated-mPD-L2-Fc were premixed with a 1:4 titration of J43v2 m2a starting at 150 nM and added to cells. Both were incubated for 1 hour on ice and cells were washed 3 times. J43v2 m2a antibody binding was detected using an anti-mouse secondary antibody (Jackson Immunoresearch, West Grove, Pa.). For the blocking experiment, biotinylated-PD-L1 or biotinylated-PD-L2 was detected using streptavidin-PE (Life Technologies, Carlsbad, Calif.). Both binding and blocking were incubated for 30 minutes on ice and were washed and read on a flow cytometer (MACSQuant).

Example 13: J43v2 m2a Induces Diabetes in NOD Mouse Model

In this Example, anti-PD-1 J43v2 m2a antibody was confirmed to induce diabetes in the NOD mouse model.

Antibody J43v2 m2a was confirmed to induce diabetes in NOD mice as follows. The NOD mice, substrain NOD/ShiLtJ were obtained from Jackson Laboratory at 8 weeks and acclimated on site for 2 weeks. At 10 weeks, mice were checked for diabetes prior to enrollment, grouped, and dosed as set forth in Table 16. FIG. 21 shows the dose-dependent induction of diabetes in NOD mice by J43v2 m2a. At day eight after a single dose, 100% of mice in the mIgG2a isotype control group remained non-diabetic, while 0%, 14% and 86% of the 20 mg/kg, 3 mg/kg and 1 mg/kg groups, respectively, remained non-diabetic.

TABLE 16 Study design Dose Dose volume Group Count Gender Treatment (mg/kg) (mL/kg) Schedule Route 1 7 F mIgG2a (C1.18.4) 20 10 q7dx1 IP 2 7 F Anti-PD-1 (J43v2) 20 10 q7dx1 IP 3 7 F Anti-PD-1 (J43v2) 5 10 q7dx1 IP 4 7 F Anti-PD-1 (J43v2) 1 10 q7dx1 IP

Example 14: Activatable Anti-Mouse PD-1 J43 Masking Moieties

This example describes identification of masking moieties (MM) that reduce binding of anti-PD-1 J43 antibody to its target.

Anti-PD-1 J43v2 mIgG2a was used to screen libraries using a method similar to that described in PCT International Publication Number WO 2010/081173, published 15 Jul. 2010. The screening consisted of one round of MACS and three rounds of FACS sorting. For the initial MACS, approximately 1×10¹² cells were incubated with J43v2 m2a antibody at a concentration of 200 nM, and approximately 9×10⁷ binders were collected using Protein-G Dynabeads (Life Technologies, Carlsbad, Calif.). FACS rounds were conducted labeling cells with DyLight 650 (Thermo-Fisher) labeled J43v2 m2a antibody and collecting cells with strongest fluorescence as follows: 200 nM J43-650 collecting brightest 3% for FACS round 1 (F1), 20 nM J43-650 collecting brightest 8% for FACS round 2 (F2) and 1 nM J43-650 collecting two populations, the brightest 20% and the brightest 0.5% for FACS round 3 (F3-20 and F3-0.5). Individual peptide clones from F2, F3-20 and F3-0.5 were identified by sequence analysis (Table 17), and selected peptide clones MP001-MP014 were subsequently verified for their ability to bind J43-650 (masking moiety MP001 is also referred to herein as MP01 and/or MP-01; masking moiety MP002 is also referred to herein as MP02 and/or MP-02, and so on).

TABLE 17 Masking moiety sequences Mask Amino Acid Sequence SEQ ID NO: MP001 DYTYCRWVNWCLSGV 384 MP002 ILCPEDPWGHKCKLP 385 MP003 TNIWSCQTYCDHKHK 386 MP004 SDHKCKLQNCMNTKV 387 MP005 PGNCHPMQKEMCQFI 388 MP006 VEHLCYTHNKCKHPD 389 MP007 TIPRCGQHPKCKDTL 390 MP008 ACRICQDHPKTKWNS 391 MP009 LIQCTGNLDHKCKHY 392 MP010 IPCHHSADHKHKCTS 393 MP011 SRQICADYNCHNKYK 394 MP012 QPCNPQIDHKIKCIY 395 MP013 HYTICMTHNKCKDMA 396 MP014 ANSCLAVEHKCKHNY 397 MP015 AALHCTEHKCKNHIK 398 MP016 APCIINTVDWKSCEI 399 MP017 ATNWCTHKQKCKQDM 400 MP018 DCYNEHKLKTRVCNN 401 MP019 DEMQCSHKQKCTNSK 402 MP020 DVGICSQHNKCKPTK 403 MP021 EKYCSSDDHKCKITL 404 MP022 ELECSHNKVKNCIQI 405 MP023 ELHPCNTHKCKPIVN 406 MP024 EVGSCNHPKCKSNNY 407 MP025 EYSPSLAHPKLKDNA 408 MP026 FESLHPKGKHPEDLG 409 MP027 FPLCVRADRVCGDAQ 410 MP028 FQAPPASHNKLKPSL 411 MP029 GAIDSCHHKCKSPHY 412 MP030 GKIYTCEHNCTFGYS 413 MP031 HCTVNNHSSDHKCKI 414 MP032 HGTQCTHNKCKPILS 415 MP033 HIGWCLHPKCKTTTT 416 MP034 HLRTCIQKWCEHKMK 417 MP035 HTDCTMMSNHKCKIN 418 MP036 IRQQCTALACLLKVH 419 MP037 KGCSTHKMRAYCNQM 420 MP038 KMFTPCKIWCNNSYN 421 MP039 KTMCSGHKQKCNNSS 422 MP040 LACHSASLVDHKCKL 423 MP041 LCNVSMDHKHKPCYL 424 MP042 LGLNCFSEHKCKEHM 425 MP043 LGTCTHKHKNCNYTL 426 MP044 LHEGCTTHNKCKPIA 427 MP045 LKRSCTGHWTCYTNW 428 MP046 LQRCTHKEKYCHAIH 429 MP047 LSHCYDHKRKCSYIV 430 MP048 LSKCHNKEKNCSNNN 431 MP049 MDTCEMHKQKCRPSF 432 MP050 MHNECLTHKCKVPIT 433 MP051 MLTLCNTNACHKEKN 434 MP052 MRPCLNNLEHKCKHY 435 MP053 MSRCPTHKMKCSLNI 436 MP054 MWICQEHKLKCMTDT 437 MP055 MYYCKRRSAFYCTLN 438 MP056 NDCQHDKQMHKCKMH 439 MP057 NFGPCPMLLGCFGFR 440 MP058 NHTDCSHPKCKSHDS 441 MP059 NLNCPHKQKNCDKYH 442 MP060 NPQCTPIDHKCKTHH 443 MP061 NTTSCTHPKCKHQGK 444 MP062 NVGGCDNYGCHKLKN 445 MP063 PCSPGNLTWDHKCKY 446 MP064 PFTKCHGFNKCKEHT 447 MP065 PGDKCTHKEKCYYNN 448 MP066 PNICNLDHKRKCRIN 449 MP067 PQLACKHPKCKDAGN 450 MP068 PSCTMWTHGGVCKHA 451 MP069 PSHRHPLAKPGFRVE 452 MP070 PTCFKTHNKSKCNRV 453 MP071 PTPVCHHNFHCFGYD 454 MP072 QATCQWKKRSKCHNK 455 MP073 QHSWCQHKAKCNYGN 456 MP074 QNCSPTYTTHKCKLT 457 MP075 QSSNCEHKRKCSSTS 458 MP076 RPCLLGLVPDHKCKL 459 MP077 RRSCMRSINTCKQKY 460 MP078 RSSCPTVTPQNCENQ 461 MP079 RTMCLDLNHKCKPSN 462 MP080 RTYWCTNHNKCKHNM 463 MP081 RVENCEHNQYCHKWK 464 MP082 SCHEDDHKNKNICSL 465 MP083 SDTCVMNHPKCKRDN 466 MP084 SSTCFHPNQKECMTK 467 MP085 SSYCGGITMRCRRAM 468 MP086 STGYCTYVNWCNYTN 469 MP087 THKCKLHLQVCTQTT 470 MP088 TMNCTHPKQKCQHTN 471 MP089 TNVLCESHNCDHKNK 472 MP090 TQHAASLGVEHKSKI 473 MP091 TQLPCFDDHKCKNTN 474 MP092 TSDSCMRQKCEHKEK 475 MP093 TTCDDHKYKHKCAQL 476 MP094 TTCGAHKEKQHCIYT 477 MP095 TTYCAYWHNKCKFET 478 MP096 VGPTCGHAKCKQSEV 479 MP097 VSHPCNTHKCKTNIV 480 MP098 WDCRNTSHPKLKCHN 481 MP099 WSPCNSDHKRKCNNG 482 MP100 YANMSCEYDCHKMKY 483 MP101 YANPCTHKEKCHFKN 484 MP102 YDCSPSWTHPKCKHK 485 MP103 YGWTCTTHPKCKTTN 486 MP104 YQKCHPKAKDCGNNT 487 MP105 YWECPNMEHNKCKNN 488 MP106 PMGNRYCVLDHPKLK 489 MP107 GHKSCCQKHCEYTQT 490 MP108 LYLEMCSCCCWESIT 491 MP109 ACQAQHCYKTYACKP 492 MP110 CCYTCSVHPKCKNQL 493 MP111 CKHRCSHKEKCPANH 494 MP112 CHVLFCLMQCCRWSL 495 MP113 LNSSLVFDHPKAKPN 496 MP114 MCLLCRSKFGCKVKG 497 MP115 IICNDHKCKQNQCNN 498 MP116 IRCSLRDSLCGCERM 499 MP117 TSCQPPKHKCTCNHG 500 MP118 TQCPHRCVKPNCWLH 501 MP119 KCCETKRNHKHCTYK 502 MP120 LPHCCHKAKHCNHTS 503 MP121 PAMCAAIHEKCCIKV 504 MP122 PRSCGNQLCPCHYYK 505 MP123 TNKCSCNHNMKCINY 506 MP124 VETCCQHNKCKYPFI 507 MP125 IFCCSNHEDHKCKTN 508 MP126 VCRLICPLTCVIGVG 509 MP127 FHGCCSVYSCLTNPG 510 MP128 ALACHPKQKPLEGQL 511 MP129 SIICCATSSCPLKHL 512 MP130 APCCRPHKEKPIDSR 513 MP131 WELCCPSADCRVAMG 514

Example 15: Characterization of Activatable Anti-Mouse PD-1 Antibodies

This example describes activatable anti-PD-1 antibodies of the disclosure with reduced binding to hPD-1 compared to binding by parental antibody.

Masking efficiencies were evaluated by standard plate ELISA. Briefly, mouse PD-1-Fc (R and D systems, Minneapolis, Minn.) was adsorbed to the wells of a 96-well ELISA plate. Purified J43v2 and activatable J43v2 antibodies comprising the indicated masks, cleavable moiety 2001, and antibody J43v2 were applied to the plate in serial dilution and allowed to bind. Bound antibody and activatable antibodies were detected with anti-mouse IgG-HRP conjugate (Sigma, St Louis, Mo.) and visualized with the chromogenic substrate TMB (Thermo Scientific, Rockford, Ill.). Plots were generated in Prizm (Sigma Plot). All activatable J43 antibodies showed strongly decreased binding compared with the parental J43 (FIG. 22).

In some embodiments, the activatable antibody also includes a spacer sequence. In some embodiments, the spacer is joined directly to the MM of the activatable antibody. In some embodiments, the spacer is joined directly to the MM of the activatable antibody in the structural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB. In some embodiments, the spacer joined directly to the N-terminus of MM of the activatable antibody is selected from the group consisting of QGQSGQG (SEQ ID NO: 362), QGQSGQ (SEQ ID NO: 913), QGQSG (SEQ ID NO: 914), QGQS (SEQ ID NO: 915), QGQ (SEQ ID NO: 916), QG (SEQ ID NO: 917), and Q. In some embodiments, the spacer includes at least the amino acid sequence QGQSGQG (SEQ ID NO: 362). In some embodiments, the spacer includes at least the amino acid sequence QGQSGQ (SEQ ID NO: 913). In some embodiments, the spacer includes at least the amino acid sequence QGQSG (SEQ ID NO: 914). In some embodiments, the spacer includes at least the amino acid sequence QGQS (SEQ ID NO: 915), In some embodiments, the spacer includes at least the amino acid sequence QGQ (SEQ ID NO: 916). In some embodiments, the spacer includes at least the amino acid sequence QG (SEQ ID NO: 917). In some embodiments, the spacer includes at least the amino acid residue Q. In some embodiments, the activatable antibody does not include a spacer sequence.

Additional examples of spacers include GQSGQG (SEQ ID NO: 2042), QSGQG (SEQ ID NO: 2043), SGQG (SEQ ID NO: 2044), GQG(SEQ ID NO: 2045), QG(SEQ ID NO: 2046), and G.

While the sequences shown below include the spacer sequence of SEQ ID NO: 362, those of ordinary skill in the art appreciate that the activatable anti-PD-1 antibodies of the disclosure can include any suitable spacer sequence, such as, for example, a spacer sequence selected from the group consisting of QGQSGQG (SEQ ID NO: 362), QGQSGQ (SEQ ID NO: 913), QGQSG (SEQ ID NO: 914), QGQS (SEQ ID NO: 915), QGQ (SEQ ID NO: 916), QG (SEQ ID NO: 917), and Q. Additional examples of spacers include GQSGQG (SEQ ID NO: 2042), QSGQG (SEQ ID NO: 2043), SGQG (SEQ ID NO: 2044), GQG(SEQ ID NO: 2045), QG(SEQ ID NO: 2046), and G. While the sequences shown below include the spacer sequence of SEQ ID NO: 362, those of ordinary skill in the art will also appreciate that activatable anti-PD-1 antibodies of the disclosure in some embodiments do not include a spacer sequence.

Activatable Anti-Mouse PD-1 Light Chain Variable Domains

[spacer (SEQ ID NO: 362)][J43 MP001 2001 (SEQ ID NO: 999)] amino acid sequence: (SEQ ID NO: 515) [QGQSGQG][DYTYCRWVNWCLSGVGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRA EDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL] J43 MP001 2001 amino acid sequence: (SEQ ID NO: 1077) DYTYCRWVNWCLSGVGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETVKITCSGD QLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFS GYVDSDSKLYVFGSGTQLTVL [spacer (SEQ ID NO: 918)][J43 MP001 2001 (SEQ ID NO: 1000)] nucleic acid sequence: (SEQ ID NO: 516) [CAAGGCCAGTCTGGCCAAGGT]GATTATACGTATTGCCGTTGGGTTAATTGGTGCTTGTCTG GGGTGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAA TCACGGCGGAGGCTCTTATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACT GTCAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGTTTCATCAAAGGT CAGACCAGACCATTTTGCAAGTGATATATGATGATAATAAGCGCCCCTCGGGTATCCCTGAAAG AATCTCTGGGTCCAGCTCAGGGACAACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGAT GAAGGTGACTATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTTGGCA GCGGAACCCAGCTCACCGTCCT [spacer (SEQ ID NO: 362)][J43 MP002 2001 (SEQ ID NO: 1001)] amino acid sequence: (SEQ ID NO: 517) [QGQSGQG][ILCPEDPWGHKCKLPGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRA EDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL] J43 MP002 2001 amino acid sequence: (SEQ ID NO: 1078) ILCPEDPWGHKCKLPGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETVKITCSGD QLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFS GYVDSDSKLYVFGSGTQLTVL [spacer (SEQ ID NO: 918)][J43 MP002 2001 (SEQ ID NO: 1002)] nucleic acid sequence: (SEQ ID NO: 518) [CAAGGCCAGTCTGGCCAAGGT][ATTCTGTGCCCGGAGGATCCGTGGGGGCATAAGTGCAAGC TGCCTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAA TCACGGCGGAGGCTCTTATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACT GTCAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGTTTCATCAAAGGT CAGACCAGACCATTTTGCAAGTGATATATGATGATAATAAGCGCCCCTCGGGTATCCCTGAAAG AATCTCTGGGTCCAGCTCAGGGACAACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGAT GAAGGTGACTATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTTGGCA GCGGAACCCAGCTCACCGTCCTA [spacer (SEQ ID NO: 362)][J43 MP003 2001 (SEQ ID NO: 1003)] amino acid sequence: (SEQ ID NO: 519) [QGQSGQG][TNIWSCQTYCDHKHKGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRA EDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL] J43 MP003 2001 amino acid sequence: (SEQ ID NO: 1079) TNIWSCQTYCDHKHKGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETVKITCSGD QLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFS GYVDSDSKLYVFGSGTQLTVL [spacer (SEQ ID NO: 918)][J43 MP003 2001 (SEQ ID NO: 1004)] nucleic acid sequence: (SEQ ID NO: 520) [CAAGGCCAGTCTGGCCAAGGT][ACGAATATTTGGAGTTGCCAGACTTATTGCGATCATAAGC ATAAGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAA TCACGGCGGAGGCTCTTATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACT GTCAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGTTTCATCAAAGGT CAGACCAGACCATTTTGCAAGTGATATATGATGATAATAAGCGCCCCTCGGGTATCCCTGAAAG AATCTCTGGGTCCAGCTCAGGGACAACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGAT GAAGGTGACTATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTTGGCA GCGGAACCCAGCTCACCGTCCTA [spacer (SEQ ID NO: 362)][J43 MP004 2001 (SEQ ID NO: 1005)] amino acid sequence: (SEQ ID NO: 521) [QGQSGQG][SDHKCKLQNCMNTKVGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRA EDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL] J43 MP004 2001 amino acid sequence: (SEQ ID NO: 1080) SDHKCKLQNCMNTKVGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETVKITCSGD QLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFS GYVDSDSKLYVFGSGTQLTVL [spacer (SEQ ID NO: 918)][J43 MP004 2001 (SEQ ID NO: 1006)] nucleic acid sequence: (SEQ ID NO: 522) [CAAGGCCAGTCTGGCCAAGGT][AGTGATCATAAGTGCAAGCTTCAGAATTGCATGAATACTA AGGTTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAA TCACGGCGGAGGCTCTTATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACT GTCAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGTTTCATCAAAGGT CAGACCAGACCATTTTGCAAGTGATATATGATGATAATAAGCGCCCCTCGGGTATCCCTGAAAG AATCTCTGGGTCCAGCTCAGGGACAACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGAT GAAGGTGACTATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTTGGCA GCGGAACCCAGCTCACCGTCCTA [spacer (SEQ ID NO: 362)][J43 MP005 2001 (SEQ ID NO: 1007)] amino acid sequence: (SEQ ID NO: 523) [QGQSGQG][PGNCHPMQKEMCQFIGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRA EDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL] J43 MP005 2001 amino acid sequence: (SEQ ID NO: 1081) PGNCHPMQKEMCQFIGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETVKITCSGD QLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFS GYVDSDSKLYVFGSGTQLTVL [spacer (SEQ ID NO: 918)][J43 MP005 2001 (SEQ ID NO: 1008)] nucleic acid sequence: (SEQ ID NO: 524) [CAAGGCCAGTCTGGCCAAGGT][CCTGGTAATTGCCATCCTATGCAGAAGGAGATGTGCCAGT TTATTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAA TCACGGCGGAGGCTCTTATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACT GTCAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGTTTCATCAAAGGT CAGACCAGACCATTTTGCAAGTGATATATGATGATAATAAGCGCCCCTCGGGTATCCCTGAAAG AATCTCTGGGTCCAGCTCAGGGACAACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGAT GAAGGTGACTATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTTGGCA GCGGAACCCAGCTCACCGTCCTA [spacer (SEQ ID NO: 362)][J43 MP006 2001 (SEQ ID NO: 1009)] amino acid sequence: (SEQ ID NO: 525) [QGQSGQG][VEHLCYTHNKCKHPDGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRA EDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL] J43 MP006 2001 amino acid sequence: (SEQ ID NO: 1082) VEHLCYTHNKCKHPDGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETVKITCSGD QLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFS GYVDSDSKLYVFGSGTQLTVL [spacer (SEQ ID NO: 918)][J43 MP006 2001 (SEQ ID NO: 1010)] nucleic acid sequence: (SEQ ID NO: 526) [CAAGGCCAGTCTGGCCAAGGT][GTTGAGCATTTGTGCTATACGCATAATAAGTGCAAGCATC CTGATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAA TCACGGCGGAGGCTCTTATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACT GTCAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGTTTCATCAAAGGT CAGACCAGACCATTTTGCAAGTGATATATGATGATAATAAGCGCCCCTCGGGTATCCCTGAAAG AATCTCTGGGTCCAGCTCAGGGACAACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGAT GAAGGTGACTATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTTGGCA GCGGAACCCAGCTCACCGTCCTA [spacer (SEQ ID NO: 362)][J43 MP007 2001 (SEQ ID NO: 1011)] amino acid sequence: (SEQ ID NO: 527) [QGQSGQG][TIPRCGQHPKCKDTLGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRA EDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL] J43 MP007 2001 amino acid sequence: (SEQ ID NO: 1083) TIPRCGQHPKCKDTLGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETVKITCSGD QLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFS GYVDSDSKLYVFGSGTQLTVL [spacer (SEQ ID NO: 918)][J43 MP007 2001 (SEQ ID NO: 1012)] nucleic acid sequence: (SEQ ID NO: 528) [CAAGGCCAGTCTGGCCAAGGT][ACTATTCCGAGGTGCGGTCAGCATCCGAAGTGCAAGGATA CTTTGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAA TCACGGCGGAGGCTCTTATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACT GTCAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGTTTCATCAAAGGT CAGACCAGACCATTTTGCAAGTGATATATGATGATAATAAGCGCCCCTCGGGTATCCCTGAAAG AATCTCTGGGTCCAGCTCAGGGACAACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGAT GAAGGTGACTATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTTGGCA GCGGAACCCAGCTCACCGTCCTA [spacer (SEQ ID NO: 362)][J43 MP008 2001 (SEQ ID NO: 1013)] amino acid sequence: (SEQ ID NO: 529) [QGQSGQG][ACRICQDHPKTKWNSGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRA EDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL] J43 MP008 2001 amino acid sequence: (SEQ ID NO: 1084) ACRICQDHPKTKWNSGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETVKITCSGD QLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFS GYVDSDSKLYVFGSGTQLTVL [spacer (SEQ ID NO: 918)][J43 MP008 2001 (SEQ ID NO: 1014)] nucleic acid sequence: (SEQ ID NO: 530) [CAAGGCCAGTCTGGCCAAGGT][GCGTGCCGTATTTGTCAGGATCATCCTAAGACGAAGTGGA ATTCTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAA TCACGGCGGAGGCTCTTATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACT GTCAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGTTTCATCAAAGGT CAGACCAGACCATTTTGCAAGTGATATATGATGATAATAAGCGCCCCTCGGGTATCCCTGAAAG AATCTCTGGGTCCAGCTCAGGGACAACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGAT GAAGGTGACTATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTTGGCA GCGGAACCCAGCTCACCGTCCTA [spacer (SEQ ID NO: 362)][J43 MP009 2001 (SEQ ID NO: 1015)] amino acid sequence: (SEQ ID NO: 531) [QGQSGQG][LIQCTGNLDHKCKHYGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRA EDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL] J43 MP009 2001 amino acid sequence: (SEQ ID NO: 1085) LIQCTGNLDHKCKHYGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETVKITCSGD QLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFS GYVDSDSKLYVFGSGTQLTVL [spacer (SEQ ID NO: 918)][J43 MP009 2001 (SEQ ID NO: 1016)] nucleic acid sequence: (SEQ ID NO: 532) [CAAGGCCAGTCTGGCCAAGGT][CTTATTCAGTGCACTGGTAATCTTGATCATAAGTGCAAGC ATTATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAA TCACGGCGGAGGCTCTTATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACT GTCAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGTTTCATCAAAGGT CAGACCAGACCATTTTGCAAGTGATATATGATGATAATAAGCGCCCCTCGGGTATCCCTGAAAG AATCTCTGGGTCCAGCTCAGGGACAACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGAT GAAGGTGACTATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTTGGCA GCGGAACCCAGCTCACCGTCCT [spacer (SEQ ID NO: 362)][J43 MP010 2001 (SEQ ID NO: 1017)] amino acid sequence: (SEQ ID NO: 533) [QGQSGQG][IPCHHSADHKHKCTSGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRA EDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL] J43 MP010 2001 amino acid sequence: (SEQ ID NO: 1086) IPCHHSADHKHKCTSGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETVKITCSGD QLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFS GYVDSDSKLYVFGSGTQLTVL [spacer (SEQ ID NO: 918)][J43 MP010 2001 (SEQ ID NO: 1018)] nucleic acid sequence: (SEQ ID NO: 534) [CAAGGCCAGTCTGGCCAAGGT][ATTCCTTGCCATCATAGTGCTGATCATAAGCATAAGTGCA CGAGTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAA TCACGGCGGAGGCTCTTATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACT GTCAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGTTTCATCAAAGGT CAGACCAGACCATTTTGCAAGTGATATATGATGATAATAAGCGCCCCTCGGGTATCCCTGAAAG AATCTCTGGGTCCAGCTCAGGGACAACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGAT GAAGGTGACTATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTTGGCA GCGGAACCCAGCTCACCGTCCTA [spacer (SEQ ID NO: 362)][J43 MP011 2001 (SEQ ID NO: 1019)] amino acid sequence: (SEQ ID NO: 535) [QGQSGQG][SRQICADYNCHNKYKGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRA EDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL] J43 MP011 2001 amino acid sequence: (SEQ ID NO: 1087) SRQICADYNCHNKYKGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETVKITCSGD QLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFS GYVDSDSKLYVFGSGTQLTVL [spacer (SEQ ID NO: 918)][J43 MP011 2001 (SEQ ID NO: 1020)] nucleic acid sequence: (SEQ ID NO: 536) [CAAGGCCAGTCTGGCCAAGGT][TCGCGGCAGATTTGCGCTGATTATAATTGCCATAATAAGT ATAAGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAA TCACGGCGGAGGCTCTTATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACT GTCAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGTTTCATCAAAGGT CAGACCAGACCATTTTGCAAGTGATATATGATGATAATAAGCGCCCCTCGGGTATCCCTGAAAG AATCTCTGGGTCCAGCTCAGGGACAACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGAT GAAGGTGACTATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTTGGCA GCGGAACCCAGCTCACCGTCCTA [spacer (SEQ ID NO: 362)][J43 MP012 2001 (SEQ ID NO: 1021)] amino acid sequence: (SEQ ID NO: 537) [QGQSGQG][QPCNPQIDHKIKCIYGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRA EDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL J43 MP012 2001 amino acid sequence: (SEQ ID NO: 1088) QPCNPQIDHKIKCIYGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETVKITCSGD QLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFS GYVDSDSKLYVFGSGTQLTVL [spacer (SEQ ID NO: 918)][J43 MP012 2001 (SEQ ID NO: 1022)] nucleic acid sequence: (SEQ ID NO: 538) [CAAGGCCAGTCTGGCCAAGGT][CAGCCTTGCAATCCGCAGATTGATCATAAGATTAAGTGCA TTTATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAA TCACGGCGGAGGCTCTTATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACT GTCAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGTTTCATCAAAGGT CAGACCAGACCATTTTGCAAGTGATATATGATGATAATAAGCGCCCCTCGGGTATCCCTGAAAG AATCTCTGGGTCCAGCTCAGGGACAACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGAT GAAGGTGACTATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTTGGCA GCGGAACCCAGCTCACCGTCCTA [spacer (SEQ ID NO: 362)][J43 MP013 2001 (SEQ ID NO: 1023)] amino acid sequence: (SEQ ID NO: 539) [QGQSGQG][HYTICMTHNKCKDMAGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRA EDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL] J43 MP013 2001 amino acid sequence: (SEQ ID NO: 1089) HYTICMTHNKCKDMAGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETVKITCSGD QLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFS GYVDSDSKLYVFGSGTQLTVL [spacer (SEQ ID NO: 918)][J43 MP013 2001 (SEQ ID NO: 1024)] nucleic acid sequence: (SEQ ID NO: 540) [CAAGGCCAGTCTGGCCAAGGT][CATTATACTATTTGCATGACGCATAATAAGTGCAAGGATA TGGCGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAA TCACGGCGGAGGCTCTTATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACT GTCAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGTTTCATCAAAGGT CAGACCAGACCATTTTGCAAGTGATATATGATGATAATAAGCGCCCCTCGGGTATCCCTGAAAG AATCTCTGGGTCCAGCTCAGGGACAACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGAT GAAGGTGACTATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTTGGCA GCGGAACCCAGCTCACCGTCCTA [spacer (SEQ ID NO: 362)][J43 MP014 2001 (SEQ ID NO: 1025)] amino acid sequence: (SEQ ID NO: 541) [QGQSGQG[]ANSCLAVEHKCKHNYGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRA EDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL] J43 MP014 2001 amino acid sequence: (SEQ ID NO: 1090) ANSCLAVEHKCKHNYGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETVKITCSGD QLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFS GYVDSDSKLYVFGSGTQLTVL [spacer (SEQ ID NO: 918)][J43 MP014 2001 (SEQ ID NO: 1026)] nucleic acid sequence: (SEQ ID NO: 542) [CAAGGCCAGTCTGGCCAAGGT][GCTAATAGTTGCCTTGCTGTTGAGCATAAGTGCAAGCATA ATTATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAA TCACGGCGGAGGCTCTTATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACT GTCAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGTTTCATCAAAGGT CAGACCAGACCATTTTGCAAGTGATATATGATGATAATAAGCGCCCCTCGGGTATCCCTGAAAG AATCTCTGGGTCCAGCTCAGGGACAACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGAT GAAGGTGACTATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTTGGCA GCGGAACCCAGCTCACCGTCCTA

Reformatted Anti-Mouse PD-1 Heavy and Light Chains:

J43v2 light chain (LC) amino acid sequence: (SEQ ID NO: 543) YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVLGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVT WKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHE GETVEKSLSPAECL J43v2 LC nucleotide sequence 1: (SEQ ID NO: 544) TATGAGCTGACTCAGCCACCTTCAGCATCAGTCAATGTAGGAGAGACTGT CAAAATCACCTGCTCTGGGGACCAATTGCCGAAATATTTTGCAGATTGGT TTCATCAAAGGTCAGACCAGACCATTTTGCAAGTGATATATGATGATAAT AAGCGCCCCTCGGGTATCCCTGAAAGAATCTCTGGGTCCAGCTCAGGGAC AACAGCCACCTTGACCATCAGAGATGTCCGGGCTGAGGATGAAGGTGACT ATTACTGTTTCTCAGGATATGTTGATAGTGATAGCAAATTGTATGTTTTT GGCAGCGGAACCCAGCTCACCGTCCTAGGTGGACCCAAGTCTTCTCCCAA AGTCACAGTGTTTCCACCTTCACCTGAGGAGCTCCGGACAAACAAAGCCA CACTGGTGTGTCTGGTTAATGACTTCTACCCGGGTTCTGCAACAGTGACC TGGAAGGCAAATGGAGCAACTATCAATGATGGGGTGAAGACTACAAAGCC TTCCAAACAGGGCCAAAACTACATGACCAGCAGCTACCTAAGTTTGACAG CAGACCAGTGGAAATCTCACAACAGGGTTTCCTGCCAAGTTACCCATGAA GGGGAAACTGTGGAGAAGAGTTTGTCCCCTGCAGAATGTCTT J43v2 LC nucleotide sequence 2: (SEQ ID NO: 545) TACGAGCTGACCCAGCCTCCTAGCGCCTCCGTGAATGTGGGCGAGACAGT GAAGATCACCTGTAGCGGCGACCAGCTGCCCAAGTACTTCGCCGACTGGT TCCACCAGCGGAGCGACCAGACAATCCTGCAAGTGATCTACGACGACAAC AAGCGGCCCAGCGGCATCCCCGAGAGAATCAGCGGAAGCAGCAGCGGCAC CACCGCCACCCTGACCATTAGAGATGTGCGGGCCGAGGACGAGGGCGACT ACTACTGCTTTAGCGGCTACGTGGACAGCGACAGCAAGCTGTACGTGTTC GGCTCCGGTACCCAGCTGACAGTGCTGGGCGGACCTAAGAGCAGCCCCAA AGTGACCGTGTTCCCCCCAAGCCCCGAGGAACTGAGGACCAACAAGGCCA CCCTCGTGTGCCTCGTGAACGACTTCTACCCTGGCAGCGCCACCGTGACC TGGAAAGCCAATGGCGCCACCATCAACGACGGCGTGAAAACCACCAAGCC CAGCAAGCAGGGCCAGAACTACATGACCAGCAGCTACCTGAGCCTGACCG CCGACCAGTGGAAGTCCCACAACAGAGTGTCCTGCCAAGTGACCCACGAG GGGGAAACCGTGGAAAAGAGCCTGAGCCCTGCCGAGTGCCTG J43v2 mIgG2a heavy chain (HC) amino acid sequence: (SEQ ID NO: 546) EVRLLESGGGLVKPEGSLKLSCVASGFTFSDYFMSWVRQAPGKGLEWVAH IYTKSYNYATYYSGSVKGRFTISRDDSRSMVYLQMNNLRTEDTATYYCTR DGSGYPSLDFWGQGTQVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLV KGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQS ITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPK IKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDY NSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAP QVYVLPPPEEEMTKKQVILTCMVTDPMPEDIYVEWINNGKTELNYKNTEP VLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG K J43v2 mIgG2a HC nucleic acid sequence: (SEQ ID NO: 547) GAGGTGCGGCTTCTGGAGTCTGGTGGAGGATTAGTGAAGCCTGAGGGGTC ACTGAAACTCTCCTGTGTGGCCTCTGGATTCACCTTCAGTGACTATTTCA TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGCTCAC ATATACACGAAAAGTTATAATTATGCAACTTATTACTCGGGTTCGGTGAA AGGCAGATTCACCATCTCCAGAGATGATTCCCGAAGCATGGTCTACCTGC AAATGAACAACCTGAGAACTGAGGACACGGCCACTTATTACTGTACAAGA GATGGAAGCGGATATCCCTCTCTGGATTTCTGGGGTCAAGGGACCCAAGT CACTGTCTCCTCAGCTAAAACAACAGCCCCATCGGTCTATCCACTGGCCC CTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTC AAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCT GTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACA CCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCC ATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAA AATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCC CAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAG ATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGT GGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGA ACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTAC AACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTG GATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAG CGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCA CAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGT CACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGG AGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCA GTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGA AAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACG AGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGT AAA

Example 16: Activatable Anti-mPD-1 Antibodies with a Range of Masking Efficiencies

This example describes the modulation of anti-mPD-1 antibody masking efficiencies by masking moiety truncation and single amino-acid substitution.

Four J43 activatable antibodies were selected to generate families of activatable anti-mPD-1 antibodies by masking moiety truncation or amino acid substitution of one or more MM residues. Table 18 lists the designed MMs. Activatable J43v2 antibodies with the designed MM and 2001 substrate were produced by transfection of 30 mL HEK293 cells and selected activatable antibodies (sequences MPtrunc ELISA activatable antibody LCs) were evaluated for masking by ELISA as described in Example 15 (FIG. 23).

In some embodiments, the activatable antibody also includes a spacer sequence. In some embodiments, the spacer is joined directly to the MM of the activatable antibody. In some embodiments, the spacer is joined directly to the MM of the activatable antibody in the structural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB. In some embodiments, the spacer joined directly to the N-terminus of MM of the activatable antibody is selected from the group consisting of QGQSGQG (SEQ ID NO: 362), QGQSGQ (SEQ ID NO: 913), QGQSG (SEQ ID NO: 914), QGQS (SEQ ID NO: 915), QGQ (SEQ ID NO: 916), QG (SEQ ID NO: 917), and Q. In some embodiments, the spacer includes at least the amino acid sequence QGQSGQG (SEQ ID NO: 362). In some embodiments, the spacer includes at least the amino acid sequence QGQSGQ (SEQ ID NO: 913). In some embodiments, the spacer includes at least the amino acid sequence QGQSG (SEQ ID NO: 914). In some embodiments, the spacer includes at least the amino acid sequence QGQS (SEQ ID NO: 915). In some embodiments, the spacer includes at least the amino acid sequence QGQ (SEQ ID NO: 916). In some embodiments, the spacer includes at least the amino acid sequence QG (SEQ ID NO: 917). In some embodiments, the spacer includes at least the amino acid residue Q. In some embodiments, the activatable antibody does not include a spacer sequence.

While the sequences shown below include the spacer sequence of SEQ ID NO: 362, those of ordinary skill in the art appreciate that the activatable anti-PD-1 antibodies of the disclosure can include any suitable spacer sequence, such as, for example, a spacer sequence selected from the group consisting of QGQSGQG (SEQ ID NO: 362), QGQSGQ (SEQ ID NO: 913), QGQSG (SEQ ID NO: 914), QGQS (SEQ ID NO: 915), QGQ (SEQ ID NO: 916), QG (SEQ ID NO: 917), and Q. While the sequences shown below include the spacer sequence of SEQ ID NO: 362, those of ordinary skill in the art will also appreciate that activatable anti-PD-1 antibodies of the disclosure in some embodiments do not include a spacer sequence.

TABLE 18 Masking moiety sequences Mask Amino Acid Sequence SEQ ID NO: MP8-1 QDHPKTKWNS 548 MP8-2 ACRICQDHPATKWNS 549 MP8-3 ACRICQDHPKTAWNS 550 MP8-4 ACRICQDAPKTKWNS 551 MP8-5 ACRICQDHAKTKWNS 552 MP8-6 DHPATKWNS 553 MP8-7 DHPKTAWNS 554 MP8-8 DAPKTKWNS 555 MP8-9 DAPATKWNS 556 MP8-10 ACRICQDHP 557 MP7-1 HPQSKDTL 558 MP7-2 HPKSQDTL 559 MP7-3 TIPRCGQHPLCLDTL 560 MP7-4 HPLSLDTL 561 MP7-5 HPASKDTL 562 MP7-6 HPKSADTL 563 MP5-1 PGNCHPLQKELCQFI 564 MP5-2 HPLQKELAQFI 565 MP5-3 HPLALELAQFI 566 MP5-4 PGNCHPLQLELCQFI 567 MP3-1 TNIWSCQTYCDHAHA 568 MP3-2 TNIWSCQTYCDHAHL 569 MP3-3 TNIWSCQTYCDHLHA 570 MP3-4 TNIWSCQTYCDHKHA 571

[Spacer (SEQ ID NO: 362)] [J43 MP5-2 2001 (SEQ ID NO: 1027)]  Amino Acid Sequence:  (SEQ ID NO: 572) [QGQSGQG][HPLQKELAQFIGGGSSGGSISSGLLSGRSDNHGGGSYE LTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNK RPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYV FGSGTQLTVL] J43 MP5-2 2001 Amino Acid Sequence:  (SEQ ID NO: 1113) HPLQKELAQFIGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVG ETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSS SGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL [Spacer (SEQ ID NO: 918)] [J43 MP5-2 2001 (SEQ ID NO: 1038)]  Nucleic Acid Sequence:  (SEQ ID NO: 573) [CAAGGCCAGTCTGGCCAAGGT][CATCCTCTGCAGAAGGAGCTGGCCCA GTTTATTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGT CCGGCAGATCCGACAATCACGGCGGAGGCTCTTACGAGCTGACCCAGCCT CCTAGCGCCTCCGTGAATGTGGGCGAGACAGTGAAGATCACCTGTAGCGG CGACCAGCTGCCCAAGTACTTCGCCGACTGGTTCCACCAGCGGAGCGACC AGACAATCCTGCAAGTGATCTACGACGACAACAAGCGGCCCAGCGGCATC CCCGAGAGAATCAGCGGAAGCAGCAGCGGCACCACCGCCACCCTGACCAT TAGAGATGTGCGGGCCGAGGACGAGGGCGACTACTACTGCTTTAGCGGCT ACGTGGACAGCGACAGCAAGCTGTACGTGTTCGGCTCCGGTACCCAGCTG ACAGTGCTGGGCGGACCTAAGAGCAGCCCCAAAGTGACCGTGTTCCCCCC AAGCCCCGAGGAACTGAGGACCAACAAGGCCACCCTCGTGTGCCTCGTGA ACGACTTCTACCCTGGCAGCGCCACCGTGACCTGGAAAGCCAATGGCGCC ACCATCAACGACGGCGTGAAAACCACCAAGCCCAGCAAGCAGGGCCAGAA CTACATGACCAGCAGCTACCTGAGCCTGACCGCCGACCAGTGGAAGTCCC ACAACAGAGTGTCCTGCCAAGTGACCCACGAGGGGGAAACCGTGGAAAAG AGCCTGAGCCCTGCCGAGTGCCTG [Spacer (SEQ ID NO: 362)] [J43 MP7-1 2001 (SEQ ID NO: 1039)]  Amino Acid Sequence:  (SEQ ID NO: 574) [QGQSGQG][HPQSKDTLGGGSSGGSISSGLLSGRSDNHGGGSYELTQPP SASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIP ERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLT VL] J43 MP7-1 2001 Amino Acid Sequence:  (SEQ ID NO: 1114) HPQSKDTLGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETV KITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGT TATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL [Spacer (SEQ ID NO: 918)] [J43 MP7-1 2001 (SEQ ID NO: 2062  Nucleic Acid Sequence:  (SEQ ID NO: 575) [CAAGGCCAGTCTGGCCAAGGT][CATCCGCAGTCTAAGGATACTTTGGG AGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGAT CCGACAATCACGGCGGAGGCTCTTACGAGCTGACCCAGCCTCCTAGCGCC TCCGTGAATGTGGGCGAGACAGTGAAGATCACCTGTAGCGGCGACCAGCT GCCCAAGTACTTCGCCGACTGGTTCCACCAGCGGAGCGACCAGACAATCC TGCAAGTGATCTACGACGACAACAAGCGGCCCAGCGGCATCCCCGAGAGA ATCAGCGGAAGCAGCAGCGGCACCACCGCCACCCTGACCATTAGAGATGT GCGGGCCGAGGACGAGGGCGACTACTACTGCTTTAGCGGCTACGTGGACA GCGACAGCAAGCTGTACGTGTTCGGCTCCGGTACCCAGCTGACAGTGCTG GGCGGACCTAAGAGCAGCCCCAAAGTGACCGTGTTCCCCCCAAGCCCCGA GGAACTGAGGACCAACAAGGCCACCCTCGTGTGCCTCGTGAACGACTTCT ACCCTGGCAGCGCCACCGTGACCTGGAAAGCCAATGGCGCCACCATCAAC GACGGCGTGAAAACCACCAAGCCCAGCAAGCAGGGCCAGAACTACATGAC CAGCAGCTACCTGAGCCTGACCGCCGACCAGTGGAAGTCCCACAACAGAG TGTCCTGCCAAGTGACCCACGAGGGGGAAACCGTGGAAAAGAGCCTGAGC CCTGCCGAGTGCCTG

MPtrunc ELISA Activatable Antibody LCs:

[Spacer (SEQ ID NO: 362)] [J43 MP7-5 2001 (SEQ ID NO: 1115)]  Amino Acid Sequence:  (SEQ ID NO: 576) [QGQSGQG][HPASKDTLGGGSSGGSISSGLLSGRSDNHGGGSYELTQPP SASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIP ERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLT VL] J43 MP7-5 2001 Amino Acid Sequence:  (SEQ ID NO: 1115) HPASKDTLGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGETV KITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGT TATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL [Spacer (SEQ ID NO: 918)] [J43 MP7-5 2001 (SEQ ID NO: 2061]  Nucleic Acid Sequence:  (SEQ ID NO: 577) [CAAGGCCAGTCTGGCCAAGGT][CATCCGGCGTCTAAGGATACTTTGGG AGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGAT CCGACAATCACGGCGGAGGCTCTTACGAGCTGACCCAGCCTCCTAGCGCC TCCGTGAATGTGGGCGAGACAGTGAAGATCACCTGTAGCGGCGACCAGCT GCCCAAGTACTTCGCCGACTGGTTCCACCAGCGGAGCGACCAGACAATCC TGCAAGTGATCTACGACGACAACAAGCGGCCCAGCGGCATCCCCGAGAGA ATCAGCGGAAGCAGCAGCGGCACCACCGCCACCCTGACCATTAGAGATGT GCGGGCCGAGGACGAGGGCGACTACTACTGCTTTAGCGGCTACGTGGACA GCGACAGCAAGCTGTACGTGTTCGGCTCCGGTACCCAGCTGACAGTGCTG GGCGGACCTAAGAGCAGCCCCAAAGTGACCGTGTTCCCCCCAAGCCCCGA GGAACTGAGGACCAACAAGGCCACCCTCGTGTGCCTCGTGAACGACTTCT ACCCTGGCAGCGCCACCGTGACCTGGAAAGCCAATGGCGCCACCATCAAC GACGGCGTGAAAACCACCAAGCCCAGCAAGCAGGGCCAGAACTACATGAC CAGCAGCTACCTGAGCCTGACCGCCGACCAGTGGAAGTCCCACAACAGAG TGTCCTGCCAAGTGACCCACGAGGGGGAAACCGTGGAAAAGAGCCTGAGC CCTGCCGAGTGCCTG [Spacer (SEQ ID NO: 362)] [J43 MP8-2 2001 (SEQ ID NO: 1032)]  Amino Acid Sequence:  (SEQ ID NO: 578) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDNHGGGS YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVL] J43 MP8-2 2001 Amino Acid Sequence:  (SEQ ID NO: 1116) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL [Spacer (SEQ ID NO: 918)] J43 MP8-2 2001 (SEQ ID  NO: 1033)] Nucleic Acid Sequence:  (SEQ ID NO: 579) [CAAGGCCAGTCTGGCCAAGGT][GCGTGCCGTATTTGTCAGGATCATCC TGCGACGAAGTGGAATTCTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGCTCTACGAGC TGACCCAGCCTCCTAGCGCCTCCGTGAATGTGGGCGAGACAGTGAAGATC ACCTGTAGCGGCGACCAGCTGCCCAAGTACTTCGCCGACTGGTTCCACCA GCGGAGCGACCAGACAATCCTGCAAGTGATCTACGACGACAACAAGCGGC CCAGCGGCATCCCCGAGAGAATCAGCGGAAGCAGCAGCGGCACCACCGCC ACCCTGACCATTAGAGATGTGCGGGCCGAGGACGAGGGCGACTACTACTG CTTTAGCGGCTACGTGGACAGCGACAGCAAGCTGTACGTGTTCGGCTCCG GTACCCAGCTGACAGTGCTGGGCGGACCTAAGAGCAGCCCCAAAGTGACC GTGTTCCCCCCAAGCCCCGAGGAACTGAGGACCAACAAGGCCACCCTCGT GTGCCTCGTGAACGACTTCTACCCTGGCAGCGCCACCGTGACCTGGAAAG CCAATGGCGCCACCATCAACGACGGCGTGAAAACCACCAAGCCCAGCAAG CAGGGCCAGAACTACATGACCAGCAGCTACCTGAGCCTGACCGCCGACCA GTGGAAGTCCCACAACAGAGTGTCCTGCCAAGTGACCCACGAGGGGGAAA CCGTGGAAAAGAGCCTGAGCCCTGCCGAGTGCCTG [Spacer (SEQ ID NO: 362)] [J43 MP8-8 2001 (SEQ ID NO: 1034)]  Amino Acid Sequence:  (SEQ ID NO: 580) [QGQSGQG][DAPKTKWNSGGGSSGGSISSGLLSGRSDNHGGGSYELTQP PSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGI PERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQL TVL] J43 MP8-8 2001 Amino Acid Sequence:  (SEQ ID NO: 1117) DAPKTKWNSGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGET VKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSG TTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL [Spacer (SEQ ID NO: 918)] J43 MP8-8 2001 (SEQ ID  NO: 1035)] Nucleic Acid Sequence:  (SEQ ID NO: 581) [CAAGGCCAGTCTGGCCAAGGT][GATGCTCCTAAGACGAAGTGGAATTC TGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCA GATCCGACAATCACGGCGGAGGCTCTTACGAGCTGACCCAGCCTCCTAGC GCCTCCGTGAATGTGGGCGAGACAGTGAAGATCACCTGTAGCGGCGACCA GCTGCCCAAGTACTTCGCCGACTGGTTCCACCAGCGGAGCGACCAGACAA TCCTGCAAGTGATCTACGACGACAACAAGCGGCCCAGCGGCATCCCCGAG AGAATCAGCGGAAGCAGCAGCGGCACCACCGCCACCCTGACCATTAGAGA TGTGCGGGCCGAGGACGAGGGCGACTACTACTGCTTTAGCGGCTACGTGG ACAGCGACAGCAAGCTGTACGTGTTCGGCTCCGGTACCCAGCTGACAGTG CTGGGCGGACCTAAGAGCAGCCCCAAAGTGACCGTGTTCCCCCCAAGCCC CGAGGAACTGAGGACCAACAAGGCCACCCTCGTGTGCCTCGTGAACGACT TCTACCCTGGCAGCGCCACCGTGACCTGGAAAGCCAATGGCGCCACCATC AACGACGGCGTGAAAACCACCAAGCCCAGCAAGCAGGGCCAGAACTACAT GACCAGCAGCTACCTGAGCCTGACCGCCGACCAGTGGAAGTCCCACAACA GAGTGTCCTGCCAAGTGACCCACGAGGGGGAAACCGTGGAAAAGAGCCTG AGCCCTGCCGAGTGCCTG [Spacer (SEQ ID NO: 362)] [J43 MP8-9 2001 (SEQ ID NO: 1036)]  Amino Acid Sequence:  (SEQ ID NO: 582) [QGQSGQG][DAPATKWNSGGGSSGGSISSGLLSGRSDNHGGGSYELTQP PSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGI PERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQL TVL] J43 MP8-9 2001 Amino Acid Sequence:  (SEQ ID NO: 1118) DAPATKWNSGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSASVNVGET VKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSG TTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL [Spacer (SEQ ID NO: 918)] [J43 MP8-9 2001 (SEQ ID NO: 1037)]  Nucleic Acid Sequence:  (SEQ ID NO: 583) [CAAGGCCAGTCTGGCCAAGGT][GATGCTCCTGCGACGAAGTGGAATTC TGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCA GATCCGACAATCACGGCGGAGGCTCTTACGAGCTGACCCAGCCTCCTAGC GCCTCCGTGAATGTGGGCGAGACAGTGAAGATCACCTGTAGCGGCGACCA GCTGCCCAAGTACTTCGCCGACTGGTTCCACCAGCGGAGCGACCAGACAA TCCTGCAAGTGATCTACGACGACAACAAGCGGCCCAGCGGCATCCCCGAG AGAATCAGCGGAAGCAGCAGCGGCACCACCGCCACCCTGACCATTAGAGA TGTGCGGGCCGAGGACGAGGGCGACTACTACTGCTTTAGCGGCTACGTGG ACAGCGACAGCAAGCTGTACGTGTTCGGCTCCGGTACCCAGCTGACAGTG CTGGGCGGACCTAAGAGCAGCCCCAAAGTGACCGTGTTCCCCCCAAGCCC CGAGGAACTGAGGACCAACAAGGCCACCCTCGTGTGCCTCGTGAACGACT TCTACCCTGGCAGCGCCACCGTGACCTGGAAAGCCAATGGCGCCACCATC AACGACGGCGTGAAAACCACCAAGCCCAGCAAGCAGGGCCAGAACTACAT GACCAGCAGCTACCTGAGCCTGACCGCCGACCAGTGGAAGTCCCACAACA GAGTGTCCTGCCAAGTGACCCACGAGGGGGAAACCGTGGAAAAGAGCCTG AGCCCTGCCGAGTGCCTG [Spacer (SEQ ID NO: 362)] [J43 MP8-2 2003 (SEQ ID NO: 1119)  Amino Acid Sequence:  (SEQ ID NO: 1120) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSANPRGGGS YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVL] J43 MP8-2 2003 Amino Acid Sequence:  (SEQ ID NO: 1119) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSANPRGGGSYELTQPPSA SVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPER ISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL (SEQ ID NO: 1122) [CAAGGCCAGTCTGGCCAAGGT][GCGTGCCGTATTTGTCAGGATCATCC TGCGACGAAGTGGAATTCTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGCCAATCCTCGTGGCGGAGGATCCTAC GAGCTGACCCAGCCTCCTAGCGCCTCCGTGAATGTGGGCGAGACAGTGAA GATCACCTGTAGCGGCGACCAGCTGCCCAAGTACTTCGCCGACTGGTTCC ACCAGCGGAGCGACCAGACAATCCTGCAAGTGATCTACGACGACAACAAG CGGCCCAGCGGCATCCCCGAGAGAATCAGCGGAAGCAGCAGCGGCACCAC CGCCACCCTGACCATTAGAGATGTGCGGGCCGAGGACGAGGGCGACTACT ACTGCTTTAGCGGCTACGTGGACAGCGACAGCAAGCTGTACGTGTTCGGC TCCGGTACCCAGCTGACAGTGCTG] [Spacer (SEQ ID NO: 362)] [J43 MP8-2 2005 (SEQ ID NO: 1123)  Amino Acid Sequence:  (SEQ ID NO: 1124) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGAVGLLAPPSGRSANPRGG GSYELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYD DNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLY VFGSGTQLTVL] J43 MP8-2 2005 Amino Acid Sequence:  (SEQ ID NO: 1123) ACRICQDHPATKWNSGGGSSGGAVGLLAPPSGRSANPRGGGSYELTQPPS ASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPE RISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTV L [Spacer (SEQ ID NO: 362)] [J43 MP8-2 2008 (SEQ ID NO: 1127)  Amino Acid Sequence:  (SEQ ID NO: 1128) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDGHGGGS YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVL] J43 MP8-2 2008 Amino Acid Sequence:  (SEQ ID NO: 1127) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDGHGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL [Spacer (SEQ ID NO: 918)] [J43 MP8-2 2008 (SEQ ID NO: 1129)  Nucleotide Sequence:  (SEQ ID NO: 1130) [CAAGGCCAGTCTGGCCAAGGT][GCGTGCCGTATTTGTCAGGATCATCC TGCGACGAAGTGGAATTCTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACCAGCACGGCGGAGGATCCTACGA GCTGACCCAGCCTCCTAGCGCCTCCGTGAATGTGGGCGAGACAGTGAAG ATCACCTGTAGCGGCGACCAGCTGCCCAAGTACTTCGCCGACTGGTTCC ACCAGCGGAGCGACCAGACAATCCTGCAAGTGATCTACGACGACAACA AGCGGCCCAGCGGCATCCCCGAGAGAATCAGCGGAAGCAGCAGCGGC ACCACCGCCACCCTGACCATTAGAGATGTGCGGGCCGAGGACGAGGG CGACTACTACTGCTTTAGCGGCTACGTGGACAGCGACAGCAAGCTGTAC GTGTTCGGCTCCGGTACCCAGCTGACAGTGCTG] [Spacer (SEQ ID NO: 362)] [J43 MP8-2 2012 (SEQ ID NO: 1131)  Amino Acid Sequence:  (SEQ ID NO: 1132) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSANPGGG SYELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDD NKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYV FGSGTQLTVL] J43 MP8-2 2012 Amino Acid Sequence:  (SEQ ID NO: 1131) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSANPGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL [Spacer (SEQ ID NO: 918)] [J43 MP8-2 2012 (SEQ ID NO: 616)  Nucleotide Sequence:  (SEQ ID NO: 1133) [CAAGGCCAGTCTGGCCAAGGT][GCGTGCCGTATTTGTCAGGATCATC CTGCGACGAAGTGGAATTCTGGAGGTGGCTCGAGCGGCGGCTCTATC TCTTCCGGACTGCTGTCCGGCAGATCCGCTAATCCCGGCGGAGGA TCCTACGAGCTGACCCAGCCTCCTAGCGCCTCCGTGAATGTGGGCGAGA CAGTGAAGATCACCTGTAGCGGCGACCAGCTGCCCAAGTACTTCGCCGA CTGGTTCCACCAGCGGAGCGACCAGACAATCCTGCAAGTGATCTACGAC GACAACAAGCGGCCCAGCGGCATCCCCGAGAGAATCAGCGGAAGCAGCA GCGGCACCACCGCCACCCTGACCATTAGAGATGTGCGGGCCGAGGACGA GGGCGACTACTACTGCTTTAGCGGCTACGTGGACAGCGACAGCAAGCTG TACGTGTTCGGCTCCGGTACCCAGCTGACAGTGCTG] [Spacer (SEQ ID NO: 362)] [J43 MP8-2 2011 (SEQ ID NO: 1134)  Amino Acid Sequence:  (SEQ ID NO: 1135) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDNP GGGSYELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTI LQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYV DSDSKLYVFGSGTQLTVL] J43 MP8-2 2011 Amino Acid Sequence:  (SEQ ID NO: 1134) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDNPGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVL [Spacer (SEQ ID NO: 918)] [J43 MP8-2 2011 (SEQ ID NO: 1136)  Nucleotide Sequence:  (SEQ ID NO: 1137) [CAAGGCCAGTCTGGCCAAGGT][GCGTGCCGTATTTGTCAGGATCA TCCTGCGACGAAGTGGAATTCTGGAGGTGGCTCGAGCGGCGGCTC TATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCCCGGCGGAGGA TCCTACGAGCTGACCCAGCCTCCTAGCGCCTCCGTGAATGTGGGCGAGA CAGTGAAGATCACCTGTAGCGGCGACCAGCTGCCCAAGTACTTCGCCGA CTGGTTCCACCAGCGGAGCGACCAGACAATCCTGCAAGTGATCTACGAC GACAACAAGCGGCCCAGCGGCATCCCCGAGAGAATCAGCGGAAGCAGCA GCGGCACCACCGCCACCCTGACCATTAGAGATGTGCGGGCCGAGGACGA GGGCGACTACTACTGCTTTAGCGGCTACGTGGACAGCGACAGCAAGCTG TACGTGTTCGGCTCCGGTACCCAGCTGACAGTGCTG]

Example 17: Activatable Anti-Mouse PD-1 J43 Antibodies Reduce Incidence of Diabetes in NOD Mice

In this Example, anti-PD-1 J43 activatable antibodies were analyzed for the ability to protect from anti-PD-1 induction of diabetes in NOD mice. The NOD mice, substrain NOD/ShiLtJ, were obtained from Jackson Laboratory at 8 weeks and acclimated on site for 2 weeks. At 10 weeks, mice were checked for diabetes prior to enrollment, grouped, and dosed as set forth in Table 4.

TABLE 4 Dosing regimen Dose Dose volume Group Count Gender Treatment (mg/kg) (mL/kg) Schedule Route 1 7 F mIgG2a (C1.18.4) 10 10 q7dx1 IP 2 7 F Anti-PD-1 J43 m2a 10 10 q7dx1 IP 3 7 F Anti-PD-1 J43 m2a 3 10 q7dx1 IP 4 7 F Anti-PD-1 J43 m2a 1 10 q7dx1 IP 5 7 F J43 MP7-1 2001 m2a 10 10 q7dx1 IP 6 7 F J43 MP7-1 2001 m2a 3 10 q7dx1 IP 7 7 F J43 MP8-2 2001 m2a 10 10 q7dx1 IP 8 7 F J43 MP8-2 2001 m2a 3 10 q7dx1 IP

FIG. 24 which plots % non-diabetic versus number of days post dose, shows that anti-PD-1 J43 antibody induced diabetes in NOD mice with increased frequency as dosage increased. At day fourteen post dose, the percentage of non-diabetic mice for the antibody-treated groups was 14%, 43% and 71% for the 20 mg/kg, 3 mg/kg and 1 mg/kg dose groups, respectively. Activatable antibodies J43 MP7-1 2001 m2a and J43 MP8-2 2001 m2a required increased doses to induce diabetes at frequencies comparable to the parental antibody. At day fourteen post dose with J43 MP7-1 2001 m2a, 57% of the 20 mg/kg group remained non-diabetic and all of the 3 mg/kg were non-diabetic. At day fourteen post dose with J43 MP8-2 2001 m2a, 71% of the 20 mg/kg group and 86% of the 3 mg/kg group remained non-diabetic.

Example 18: Evaluation of Efficiency of Masking Moieties

This example describes additional activatable anti-PD-1 antibodies that exhibit reduced binding to hPD-1.

Examples of additional activatable antibodies of the disclosure comprising anti-PD1 antibody A1.5 and a variety of mask and substrate combinations were produced using techniques as described herein. The amino acid and nucleic acid sequences of these activatable anti-PD-1 antibody variable domains of the disclosure are provided below. Antibodies were produced as hIgG4 containing a single amino acid substitution, S228P (Angal, et al. 1993. Mol Immunol 30:105-8.) HC and hK LC format.

Masking efficiencies of several of these activatable antibodies were determined as described herein. The results are shown in FIGS. 25A and 25B.

In some embodiments, the activatable antibody also includes a spacer sequence. In some embodiments, the spacer is joined directly to the MM of the activatable antibody. In some embodiments, the spacer is joined directly to the MM of the activatable antibody in the structural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB. In some embodiments, the spacer joined directly to the N-terminus of MM of the activatable antibody is selected from the group consisting of QGQSGQG (SEQ ID NO: 362), QGQSGQ (SEQ ID NO: 913), QGQSG (SEQ ID NO: 914), QGQS (SEQ ID NO: 915), QGQ (SEQ ID NO: 916), QG (SEQ ID NO: 917), and Q. In some embodiments, the spacer includes at least the amino acid sequence QGQSGQG (SEQ ID NO: 362). In some embodiments, the spacer includes at least the amino acid sequence QGQSGQ (SEQ ID NO: 913). In some embodiments, the spacer includes at least the amino acid sequence QGQSG (SEQ ID NO: 914). In some embodiments, the spacer includes at least the amino acid sequence QGQS (SEQ ID NO: 915), In some embodiments, the spacer includes at least the amino acid sequence QGQ (SEQ ID NO: 916). In some embodiments, the spacer includes at least the amino acid sequence QG (SEQ ID NO: 917). In some embodiments, the spacer includes at least the amino acid residue Q. In some embodiments, the activatable antibody does not include a spacer sequence. Additional examples of spacers include GQSGQG (SEQ ID NO: 2042), QSGQG (SEQ ID NO: 2043), SGQG (SEQ ID NO: 2044), GQG(SEQ ID NO: 2045), QG(SEQ ID NO: 2046), and G.

While the sequences shown below include the spacer sequence of SEQ ID NO: 362, those of ordinary skill in the art appreciate that the activatable anti-PD-1 antibodies of the disclosure can include any suitable spacer sequence, such as, for example, a spacer sequence selected from the group consisting of QGQSGQG (SEQ ID NO: 362), QGQSGQ (SEQ ID NO: 913), QGQSG (SEQ ID NO: 914), QGQS (SEQ ID NO: 915), QGQ (SEQ ID NO: 916), QG (SEQ ID NO: 917), and Q. Additional examples of spacers include GQSGQG (SEQ ID NO: 2042), QSGQG (SEQ ID NO: 2043), SGQG (SEQ ID NO: 2044), GQG(SEQ ID NO: 2045), QG(SEQ ID NO: 2046), and G While the sequences shown below include the spacer sequence of SEQ ID NO: 362, those of ordinary skill in the art will also appreciate that activatable anti-PD-1 antibodies of the disclosure in some embodiments do not include a spacer sequence.

[Spacer (SEQ ID NO: 362)] [PD1 1.5 PD01 2003 (SEQ ID NO: 1138)  Amino Acid Sequence:  (SEQ ID NO: 1139) [QGQSGQG][AMSGCSWSAFCPYLAGGGSSGGSISSGLLSGRSANPR GGGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQ QKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATY YCQQSKDVPWTFGQGTKLEIK] PD1 1.5 PD01 2003 Amino Acid Sequence:  (SEQ ID NO: 1138) AMSGCSWSAFCPYLAGGGSSGGSISSGLLSGRSANPRGGGSDIQLTQSPS SLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQG SGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLE IK [Spacer (SEQ ID NO: 918)] [PD1 1.5 PD001 2003 (SEQ ID NO: 1141)]  Nucleotide Sequence:  (SEQ ID NO: 1142) [CAAGGCCAGTCTGGCCAAGGT][GCGATGAGTGGGTGCTCGTGGT CTGCTTTTTGCCCGTATTTGGCGGGAGGTGGCTCGAGCGGCGGCTC TATCTCTTCCGGACTGCTGTCCGGCAGATCCGCCAATCCTCGTGGCGGA GGATCCGATATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCG TGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGC TTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCC CCCAAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAA GCAGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAG CAGCATGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAG GACGTGCCCTGGACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD01 2012 (SEQ ID NO: 1144)  Amino Acid Sequence:  (SEQ ID NO: 1143) [QGQSGQG][AMSGCSWSAFCPYLGGGSSGGSISSGLLSGRSANPGG GSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQK PGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYC QQSKDVPWTFGQGTKLEIK] PD1 1.5 PD01 2012 Amino Acid Sequence:  (SEQ ID NO: 1144) AMSGCSWSAFCPYLGGGSSGGSISSGLLSGRSANPGGGSDIQLTQSPSSL SASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSG VPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK [Spacer (SEQ ID NO: 918)] [PD1 1.5 PD01 2012 (SEQ ID NO: 1145)]  Nucleotide Sequence:  (SEQ ID NO: 1146) [CAAGGCCAGTCTGGCCAAGGT][GCGATGAGTGGGTGCTCGTGGTC TGCTTTTTGCCCGTATTTGGCGGGAGGTGGCTCGAGCGGCGGCTC TATCTCTTCCGGACTGCTGTCCGGCAGATCCGCTAATCCCGGCGGAGGA TCCGATATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGG GCGACAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTA CGGCATCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCC AAGCTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCA GATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAG CATGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGAC GTGCCCTGGACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD01 2011 (SEQ ID NO: 1148)  Amino Acid Sequence:  (SEQ ID NO: 1147) [QGQSGQG][AMSGCSWSAFCPYLGGGSSGGSISSGLLSGRSDNPG GGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQK PGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYC QQSKDVPWTFGQGTKLEIK] PD1 1.5 PD01 2011 Amino Acid Sequence:  (SEQ ID NO: 1148) AMSGCSWSAFCPYLGGGSSGGSISSGLLSGRSDNPGGGSDIQLTQSPSSL SASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGSG VPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEIK [Spacer (SEQ ID NO: 918)] [PD1 1.5 PD01 2011 (SEQ ID NO: 1149)]  Nucleotide Sequence:  (SEQ ID NO: 1150) [CAAGGCCAGTCTGGCCAAGGT][GCGATGAGTGGGTGCTCGTGGTCTGC TTTTTGCCCGTATTTGGCGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCCCGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD34 2001 (SEQ ID NO: 1152)  Amino Acid Sequence:  (SEQ ID NO: 1151) [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDNHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD1 1.5 PD34 2001 Amino Acid Sequence:  (SEQ ID NO: 1152) TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDNHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 1125)] [PD1 1.5 PD034 2001 (SEQ ID NO: 1153)]  Nucleotide Sequence:  (SEQ ID NO: 1154) [CAGGGGCAATCTGGCCAGGGG][ACGTCTTACTGCAGTATTGAGCATTA CCCCTGCAATACACATCATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD34 1004/GG/0001 (SEQ ID NO: 1156)  Amino Acid  Sequence:  (SEQ ID NO: 1155) [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSAVGLLAPPGGLSGRSDN HGGGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPG KAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQS KDVPWTFGQGTKLEIK] PD1 1.5 PD34 1004/GG/0001 Amino Acid Sequence:  (SEQ ID NO: 1156) TSYCSIEHYPCNTHHGGGSSGGSAVGLLAPPGGLSGRSDNHGGGSDIQLT QSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAA SNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQG TKLEIK [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD34 2005 (SEQ ID NO: 1160)  Amino Acid Sequence:  (SEQ ID NO: 1159) [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSAVGLLAPPSGRSANPRG GGGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGK APKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSK DVPWTFGQGTKLEIK] PD1 1.5 PD34 2005 Amino Acid Sequence:  (SEQ ID NO: 1160) TSYCSIEHYPCNTHHGGGSSGGSAVGLLAPPSGRSANPRGGGGSDIQLTQ SPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAAS NQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGT KLEIK [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD34 2010 (SEQ ID NO: 1164)  Amino Acid Sequence:  (SEQ ID NO: 1163) [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDYHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD1 1.5 PD34 2010 Amino Acid Sequence:  (SEQ ID NO: 1164) TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDYHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD34 2014 (SEQ ID NO: 1168)  Amino Acid Sequence:  (SEQ ID NO: 1167) [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDNIGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD1 1.5 PD34 2014 Amino Acid Sequence:  (SEQ ID NO: 1168) TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDNIGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD34 2003 (SEQ ID NO: 1170)  Amino Acid Sequence:  (SEQ ID NO: 1171) [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSANPRGGG SDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPK LLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVP WTFGQGTKLEIK] PD1 1.5 PD34 2003 Amino Acid Sequence:  (SEQ ID NO: 1170) TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSANPRGGGSDIQLTQSPS SLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQG SGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLE IK [Spacer (SEQ ID NO: 1125)] [PD1 1.5 PD034 2003 (SEQ ID NO: 1172)]  Nucleotide Sequence:  (SEQ ID NO: 1173) [CAGGGGCAATCTGGCCAGGGG][ACGTCTTACTGCAGTATTGAGCATTA CCCCTGCAATACACATCATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGCCAATCCTCGTGGCGGAGGATCCGAT ATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAG AGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCA GCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTG ATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGG CTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCG AGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACC TTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD34 2006 (SEQ ID NO: 1174)  Amino Acid Sequence:  (SEQ ID NO: 1175) [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDDHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD1 1.5 PD34 2006 Amino Acid Sequence:  (SEQ ID NO: 1174) TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDDHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 1125)] [PD1 1.5 PD034 2006 (SEQ ID NO: 1176)]  Nucleotide Sequence:  (SEQ ID NO: 1177) [CAGGGGCAATCTGGCCAGGGG][ACGTCTTACTGCAGTATTGAGCATTA CCCCTGCAATACACATCATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACGATCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD34 2007 (SEQ ID NO: 1178)  Amino Acid Sequence:  (SEQ ID NO: 1179) [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDIHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD1 1.5 PD34 2007 Amino Acid Sequence:  (SEQ ID NO: 1178) TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDIHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 1125)] [PD1 1.5 PD034 2007 (SEQ ID NO: 1180)]  Nucleotide Sequence:  (SEQ ID NO: 1181) [CAGGGGCAATCTGGCCAGGGG][ACGTCTTACTGCAGTATTGAGCATTA CCCCTGCAATACACATCATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACATTCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD34 2009 (SEQ ID NO: 1182)  Amino Acid Sequence:  (SEQ ID NO: 1183) [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDTHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] [PD1 1.5 PD34 2009 Amino Acid Sequence:  (SEQ ID NO: 1182) TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDTHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 1125)] [PD1 1.5 PD034 2009 (SEQ ID NO: 1184)]  Nucleotide Sequence:  (SEQ ID NO: 1185) [CAGGGGCAATCTGGCCAGGGG][ACGTCTTACTGCAGTATTGAGCATTA CCCCTGCAATACACATCATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACACTCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD34 2008 (SEQ ID NO: 1186)  Amino Acid Sequence:  (SEQ ID NO: 1187) [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDQHGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD1 1.5 PD34 2008 Amino Acid Sequence:  (SEQ ID NO: 1186) TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDQHGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 1125)] [PD1 1.5 PD034 2008 (SEQ ID NO: 1188)]  Nucleotide Sequence:  (SEQ ID NO: 1189) [CAGGGGCAATCTGGCCAGGGG][ACGTCTTACTGCAGTATTGAGCATTA CCCCTGCAATACACATCATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACCAGCACGGCGGAGGATCCGATATC CAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGT GACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCATCAGCT TCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTCCGGCTC TGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGCCCGAGG ACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGGACCTTT GGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD34 2012 (SEQ ID NO: 1190)  Amino Acid Sequence:  (SEQ ID NO: 1191) [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSANPGGGS DIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKL LIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPW TFGQGTKLEIK] PD1 1.5 PD34 2012 Amino Acid Sequence:  (SEQ ID NO: 1190) TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSANPGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 1125)] [PD1 1.5 PD034 2012 (SEQ ID NO: 1192)]  Nucleotide Sequence:  (SEQ ID NO: 1193) [CAGGGGCAATCTGGCCAGGGG][ACGTCTTACTGCAGTATTGAGCAT TACCCCTGCAATACACATCATGGAGGTGGCTCGAGCGGCGGCTCTATCT CTTCCGGACTGCTGTCCGGCAGATCCGCTAATCCCGGCGGAGGATCCG ATATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGA CAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCA TCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTC CGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGC CCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGG ACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD34 2013 (SEQ ID NO: 1194)  Amino Acid Sequence:  (SEQ ID NO: 1195) [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSANIG GGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGK APKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQS KDVPWTFGQGTKLEIK] PD1 1.5 PD34 2013 Amino Acid Sequence:  (SEQ ID NO: 1194) TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSANIGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 1125)] [PD1 1.5 PD034 2013 (SEQ ID NO: 1196)]  Nucleotide Sequence:  (SEQ ID NO: 1197) [CAGGGGCAATCTGGCCAGGGG][ACGTCTTACTGCAGTATTGAGCA TTACCCCTGCAATACACATCATGGAGGTGGCTCGAGCGGCGGCTCTATC TCTTCCGGACTGCTGTCCGGCAGATCCGCTAATATTGGCGGAGGATCCG ATATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGA CAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCA TCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTC CGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGC CCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGG ACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD34 2011 (SEQ ID NO: 1198)  Amino Acid Sequence:  (SEQ ID NO: 1199) [QGQSGQG][TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDNPG GGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGK APKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQS KDVPWTFGQGTKLEIK] PD1 1.5 PD34 2011 Amino Acid Sequence:  (SEQ ID NO: 1198) TSYCSIEHYPCNTHHGGGSSGGSISSGLLSGRSDNPGGGSDIQLTQSPSS LSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQGS GVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLEI K [Spacer (SEQ ID NO: 1125)] [PD1 1.5 PD034 2011 (SEQ ID NO: 1200)]  Nucleotide Sequence:  (SEQ ID NO: 1201) [CAGGGGCAATCTGGCCAGGGG][ACGTCTTACTGCAGTATTGAGCAT TACCCCTGCAATACACATCATGGAGGTGGCTCGAGCGGCGGCTCTATCT CTTCCGGACTGCTGTCCGGCAGATCCGACAATCCCGGCGGAGGATCCGA TATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGCGAC AGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACGGCA TCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATTTTC CGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGCAGC CCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCCTGG ACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PD1 1.5 PD006 2003 (SEQ ID NO: 1202)  Amino Acid Sequence:  (SEQ ID NO: 1203) [QGQSGQG][APRCYMFASYCKSQYGGGSSGGSISSGLLSGRSANPRG GGSDIQLTQSPSSLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGK APKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQS KDVPWTFGQGTKLEIK] PD1 1.5 PD006 2003 Amino Acid Sequence:  (SEQ ID NO: 1202) APRCYMFASYCKSQYGGGSSGGSISSGLLSGRSANPRGGGSDIQLTQSPS SLSASVGDRVTITCRASESVDAYGISFMNWFQQKPGKAPKLLIYAASNQG SGVPSRFSGSGSGTDFTLTISSMQPEDFATYYCQQSKDVPWTFGQGTKLE IK [Spacer (SEQ ID NO: 918)] [PD1 1.5 PD006 2003 (SEQ ID NO: 1204)]  Nucleotide Sequence:  (SEQ ID NO: 1205) [CAAGGCCAGTCTGGCCAAGGT][GCGCCTAGGTGCTATATGTTTGCG TCGTATTGCAAGAGTCAGTATGGAGGTGGCTCGAGCGGCGGCTCTATCT CTTCCGGACTGCTGTCCGGCAGATCCGCCAATCCTCGTGGCGGAGGATC CGATATCCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGC GACAGAGTGACCATCACCTGTAGAGCCAGCGAGAGCGTGGACGCTTACG GCATCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAG CTGCTGATCTACGCCGCCAGCAATCAGGGCAGCGGCGTGCCAAGCAGATT TTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCATGC AGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCAAGGACGTGCCC TGGACCTTTGGCCAGGGTACCAAGCTGGAAATCAAG]

Example 19: Activatable Anti-PD1 Nivolumab Masking Moieties

This example describes identification of masking moieties (MM) that reduce binding of anti-PD1 Nivolumab antibody to its target.

Anti-PD1 Nivolumab (NV1) (see, e.g., U.S. Pat. No. 8,008,449) was used to screen libraries using a method similar to that described in PCT International Publication Number WO 2010/081173, published 15 Jul. 2010. The screening consisted of one round of MACS and four rounds of FACS sorting. For the initial MACS, approximately 6×10¹¹ cells were incubated with NV1 antibody at a concentration of 100 nM, and approximately 3×10⁷ binders were collected using Protein-A Dynabeads (Life Technologies, Carlsbad, Calif.). FACS rounds were conducted labeling cells with DyLight 650 (Thermo-Fisher) labeled NV1 antibody and collecting cells with strongest fluorescence as follows: 20 nM NV1-650 collecting brightest 13% for FACS round 1 (F1), 2 nM NV1-650 collecting brightest 2% for FACS round 2 (F2-1), 1 nM NV1-650 collecting brightest 1% for FACS round 3 (F3-1b) and 1 nM NV1-650 collecting two populations, the brightest 88% and the brightest 1% for FACS round 4 (F4-1b.1 and F4-1b.2). Individual peptide clones from F4-1b.1 and F4-1b.2 were identified by sequence analysis (Table 19), and selected peptide clones NV01-NV12 were subsequently verified for their ability to bind NV1 (masking moiety NV001 is also referred to herein as NV01 and/or NV-01; masking moiety NV002 is also referred to herein as NV02 and/or NV-02, and so on).

TABLE 19 Mask Amino Acid Sequence SEQ ID NO: NV01 RYCHAANPDRFCGIY 1206 NV02 PRVCSTDGGDYCLLP 1207 NV03 PRPQCHHRHNCPDHP 1208 NV04 KCSRPAHQNPDRCSR 1209 NV05 ASYRCPDYKCSHTKH 1210 NV06 LPRCPDHPIKCIETK 1211 NV07 YTFGCPDRYCDRAAT 1212 NV08 RGCPDFNPPSHCYTA 1213 NV09 RDYCGPQSPDYCHEI 1214 NV10 PNKPCPDLQCYVTNY 1215 NV11 PRVACGEPDLCYSNT 1216 NV12 RGCKKHTISTLTCPD 1217 NV13 PAYRCPDRPPCKNQM 1218 NV14 NARCYPYFGDNCHMN 1220 NV15 PTLRCPDRWCYDSPR 1221 NV16 PSNLCPDKWCQTWRS 1222 NV17 TPRYCAASYCPAHGY 1223 NV18 RPGCGAVSPRCPDAP 1224 NV19 VLRCHKQNPDNCNNH 1225 NV20 GVKSCREPDFCSRGS 1226 NV21 RNNLCPDYSCNNHNS 1227 NV22 RAACHRLNPDACTNG 1228 NV23 VCQSDRIPDYVTCTD 1229 NV24 RNCRIASINPDYCNI 1230 NV25 KEWRCPDYKCKPSYH 1231 NV26 NLRICHKSLCPDYIK 1232 NV27 NTHKCSNTNICPSFN 1233 NV28 STRYCQASQCQMSPY 1234 NV29 THRFCTASLCNKNTS 1235 NV30 YTLCNTRSPDWCPNK 1236 NV31 IRCTTGQSPDYCPQS 1237 NV32 RCNQPDKNDQMLCNI 1238 NV33 GTCRTDHQSPDYCYY 1239 NV34 RGCFRSGDSLGMCPD 1240 NV35 SGCFDSNEHRHCSRI 1241 NV36 NRCMKLWYNPDCVAR 1242 NV37 PLCARPHYWSPCDQS 1243 NV38 DSKCHPNSPDYCFNS 1244 NV39 NGSCRPLGGDFCGNR 1245 NV40 KTRCIEMSGDYCAKS 1246 NV41 IRPCMYNWGDLCNQF 1247 NV42 VKTCMENNPDYCYNN 1248 NV43 LRMCFEASGDYCDQQ 1249 NV44 IRKCQLDGPDQCMLT 1250 NV45 KWKCHKNNPNYCNNR 1251 NV46 RTMCLDTNPDYCQSH 1252 NV47 LAACHSMDSHRCPDY 1253 NV48 RSPCIHNATMCPDYT 1254 NV49 MPRCPDWPPRCSMVI 1255 NV50 VRQLCRLPDYCPSGK 1256 NV51 PRPPCAQSLNCPDRA 1257 NV52 SFGRCTLVRTCPDFM 1258 NV53 RDKPCPDFSCATIHY 1259 NV54 ATKPCPDRWCTMSTL 1260 NV55 SSNRCPDLRCTHHNM 1261 NV56 RGSMCPDLHCSLSHI 1262 NV57 NYQRCPDRTCMHNII 1263 NV58 QKRPCPDRKCHAHYN 1264 NV59 QNHRCPDRWCNKTTN 1265 NV60 RLNLCPDKHCHMTNL 1266 NV61 PQDRCPDKRCTNPGN 1267 NV62 SRWRCPDYKCEHGKY 1268 NV63 YENQCPDLYCNRYSM 1269 NV64 TARSCPVFNCPDNNS 1270 NV65 MDQRCPDAWCTSKPK 1271 NV66 GDLRCPDRLCPRHSL 1272 NV67 IQYLCPDYHCKASNN 1273 NV68 QHHRCPDRYCNSNNN 1274 NV69 TVALCPDYSCYHINN 1275 NV70 SPWRCPDRYCLSNHD 1276 NV71 SSKRCPDRFCNKTHA 1277 NV72 HTDRCPDYKCSQNHF 1278 NV73 SRSNCTPQRCNSDYH 1279 NV74 FAARCPDKYCAIHTN 1280 NV75 GSARCPDLVCQQTKQ 1281 NV76 RNLMCPDKFCNKNTK 1282 NV77 NIRLCPDKVCTPTWV 1283 NV78 MTDLCPDAHCAKTHM 1284 NV79 PYSRLCAYPCPDFVG 1285 NV80 LCGCARSPDYCKCRG 1286 NV81 WGRCERVPDCCCPRG 1287 NV82 TRNTCHTRICYGMAC 1288 NV83 CVCTSCSSYWTLCPD 1289 NV84 LCCSRGSNCPDRCTW 1290 NV85 CCPLCQANMCPDNQS 1291 NV86 ECKLCCPDLYCGGTM 1292 NV87 CSNPMCAYCCPDLIL 1293 NV88 CPRCNTYSKHDCYHQ 1294 NV89 FCCASKMPAPSNCHT 1295

Example 20: Activatable Anti-PD1 Nivolumab Antibodies

This example describes examples of activatable anti-PD1 Nivolumab antibodies of the disclosure.

Activatable anti-PD1 NV1 antibodies comprising an anti-PD1 NV1 masking moiety, a cleavable moiety, and an anti-PD1 NV1 antibody of the disclosure were produced according to methods similar to those described in PCT Publication Nos. WO 2009/025846 and WO 2010/081173. One embodiment of the disclosure is an anti-PD-1 activatable antibody comprising a heavy chain comprising amino acid sequence SEQ ID NO: 1346. One embodiment of the disclosure is an anti-PD-1 activatable antibody comprising a light chain comprising amino acid sequence SEQ ID NO: 626. One embodiment of the disclosure is an anti-PD-1 activatable antibody comprising a heavy chain comprising amino acid sequence SEQ ID NO: 1346 and a light chain comprising amino acid sequence SEQ ID NO: 626. One embodiment of the disclosure is an anti-PD-1 activatable antibody comprising the CDRs of an anti-PD-1 activatable antibody comprising a heavy chain comprising amino acid sequence SEQ ID NO: 1346 and a light chain comprising amino acid sequence SEQ ID NO: 626.

The amino acid and nucleic acid sequences of several activatable anti-PD1 antibody variable domains of the disclosure are provided below. Antibodies were produced as hIgG4 containing a single amino acid substitution, S228P (Angal, et al. 1993. Mol Immunol 30:105-8.) HC and hK LC format.

NV1 Light Chain Sequence:  (SEQ ID NO: 626) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTF GQGTKVEIK [Spacer (SEQ ID NO: 362)] [NV1 NV01 2001 (SEQ ID NO: 1296) Amino Acid Sequence:  (SEQ ID NO: 1297) [QGQSGQG][RYCHAANPDRFCGIYGGGSSGGSISSGLLSGRSDNHGG SEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLI YDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRT FGQGTKVEIK] NV1 NV01 2001 Amino Acid Sequence:  (SEQ ID NO: 1296) RYCHAANPDRFCGIYGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK [Spacer (SEQ ID NO: 918)] [NV1 NV01 2001 (SEQ ID NO: 1298)]  Nucleotide Sequence:  (SEQ ID NO: 1299) [CAAGGCCAGTCTGGCCAAGGT][CGGTATTGCCATGCTGCGAATCCT GATCGGTTTTGCGGTATTTATGGAGGTGGCTCGAGCGGCGGCTCTATCT CTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGAT CGTGCTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGA GCCACCCTGAGCTGTAGAGCCAGCCAGAGCGTGTCCAGCTACCTGGCCT GGTATCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTACGATGCC AGCAATAGAGCCACCGGCATCCCCGCCAGATTTTCCGGCTCTGGCAGCGG CACCGACTTCACCCTGACCATCAGCTCCCTGGAACCCGAGGACTTCGCCG TGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGGACATTTGGCCAGGGC ACCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [NV1 NV02 2001 (SEQ ID NO: 1300) Amino Acid Sequence:  (SEQ ID NO: 1301) [QGQSGQG][PRVCSTDGGDYCLLPGGGSSGGSISSGLLSGRSD NHGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPR LLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWP RTFGQGTKVEIK] NV1 NV02 2001 Amino Acid Sequence:  (SEQ ID NO: 1300) PRVCSTDGGDYCLLPGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK [Spacer (SEQ ID NO: 918)] [NV1 NV02 2001 (SEQ ID NO: 1302)]  Nucleotide Sequence:  (SEQ ID NO: 1303) [CAAGGCCAGTCTGGCCAAGGT][CCTAGGGTTTGCTCTACTGA TGGTGGTGATTATTGCTTGCTTCCTGGAGGTGGCTCGAGCGGCGGCTCTA TCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAG ATCGTGCTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAG AGCCACCCTGAGCTGTAGAGCCAGCCAGAGCGTGTCCAGCTACCTGGCCT GGTATCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTACGATGCC AGCAATAGAGCCACCGGCATCCCCGCCAGATTTTCCGGCTCTGGCAGCGG CACCGACTTCACCCTGACCATCAGCTCCCTGGAACCCGAGGACTTCGCCG TGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGGACATTTGGCCAGGGC ACCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [NV1 NV03 2001 (SEQ ID NO: 1304) Amino Acid Sequence:  (SEQ ID NO: 1305) [QGQSGQG][PRPQCHHRHNCPDHPGGGSSGGSISSGLLSGRSD NHGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPR LLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWP RTFGQGTKVEIK] (SEQ ID NO: 1304) PRPQCHHRHNCPDHPGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK [Spacer (SEQ ID NO: 918)] [NV1 NV03 2001 (SEQ ID NO: 1306)]  Nucleotide Sequence:  (SEQ ID NO: 1307) [CAAGGCCAGTCTGGCCAAGGT][CCTCGTCCGCAGTGCCATCA TCGGCATAATTGTCCTGATCATCCTGGAGGTGGCTCGAGCGGCGGCTCTA TCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAG ATCGTGCTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAG AGCCACCCTGAGCTGTAGAGCCAGCCAGAGCGTGTCCAGCTACCTGGCCT GGTATCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTACGATGCC AGCAATAGAGCCACCGGCATCCCCGCCAGATTTTCCGGCTCTGGCAGCGG CACCGACTTCACCCTGACCATCAGCTCCCTGGAACCCGAGGACTTCGCCG TGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGGACATTTGGCCAGGGC ACCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [NV1 NV04 2001 (SEQ ID NO: 1308) Amino Acid Sequence:  (SEQ ID NO: 1309) [QGQSGQG][KCSRPAHQNPDRCSRGGGSSGGSISSGLLSGRSD NHGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPR LLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWP RTFGQGTKVEIK] NV1 NV04 2001 Amino Acid Sequence:  (SEQ ID NO: 1308) KCSRPAHQNPDRCSRGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK [Spacer (SEQ ID NO: 918)] [NV1 NV04 2001 (SEQ ID NO: 1310)]  Nucleotide Sequence:  (SEQ ID NO: 1311) [CAAGGCCAGTCTGGCCAAGGT][AAGTGCTCGCGGCCTGCTCA TCAGAATCCGGATCGTTGCTCGCGAGGAGGTGGCTCGAGCGGCGGCTCTA TCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAG ATCGTGCTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAG AGCCACCCTGAGCTGTAGAGCCAGCCAGAGCGTGTCCAGCTACCTGGCCT GGTATCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTACGATGCC AGCAATAGAGCCACCGGCATCCCCGCCAGATTTTCCGGCTCTGGCAGCGG CACCGACTTCACCCTGACCATCAGCTCCCTGGAACCCGAGGACTTCGCCG TGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGGACATTTGGCCAGGGC ACCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [NV1 NV05 2001 (SEQ ID NO: 1312) Amino Acid Sequence:  (SEQ ID NO: 1313) [QGQSGQG][ASYRCPDYKCSHTKHGGGSSGGSISSGLLSGRSD NHGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPR LLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWP RTFGQGTKVEIK] NV1 NV05 2001 Amino Acid Sequence:  (SEQ ID NO: 1312) ASYRCPDYKCSHTKHGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK [Spacer (SEQ ID NO: 918)] [NV1 NV05 2001 (SEQ ID NO: 1314)]  Nucleotide Sequence:  (SEQ ID NO: 1315) [CAAGGCCAGTCTGGCCAAGGT][GCTTCGTATCGGTGCCCTGA TTATAAGTGCAGTCATACTAAGCATGGAGGTGGCTCGAGCGGCGGCTCTA TCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAG ATCGTGCTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAG AGCCACCCTGAGCTGTAGAGCCAGCCAGAGCGTGTCCAGCTACCTGGCCT GGTATCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTACGATGCC AGCAATAGAGCCACCGGCATCCCCGCCAGATTTTCCGGCTCTGGCAGCGG CACCGACTTCACCCTGACCATCAGCTCCCTGGAACCCGAGGACTTCGCCG TGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGGACATTTGGCCAGGGC ACCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [NV1 NV06 2001 (SEQ ID NO: 1316) Amino Acid Sequence:  (SEQ ID NO: 1317) [QGQSGQG][LPRCPDHPIKCIETKGGGSSGGSISSGLLSGRSD NHGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPR LLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWP RTFGQGTKVEIK] NV1 NV06 2001 Amino Acid Sequence:  (SEQ ID NO: 1316) LPRCPDHPIKCIETKGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK [Spacer (SEQ ID NO: 918)] [NV1 NV06 2001 (SEQ ID NO: 1318)]  Nucleotide Sequence:  (SEQ ID NO: 1319) [CAAGGCCAGTCTGGCCAAGGT][TTGCCGAGGTGCCCGGATCA TCCGATTAAGTGCATTGAGACTAAGGGAGGTGGCTCGAGCGGCGGCTCTA TCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAG ATCGTGCTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAG AGCCACCCTGAGCTGTAGAGCCAGCCAGAGCGTGTCCAGCTACCTGGCCT GGTATCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTACGATGCC AGCAATAGAGCCACCGGCATCCCCGCCAGATTTTCCGGCTCTGGCAGCGG CACCGACTTCACCCTGACCATCAGCTCCCTGGAACCCGAGGACTTCGCCG TGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGGACATTTGGCCAGGGC ACCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [NV1 NV07 2001 (SEQ ID NO: 1320) Amino Acid Sequence:  (SEQ ID NO: 1321) [QGQSGQG][YTFGCPDRYCDRAATGGGSSGGSISSGLLSGRSDNHGGSE IVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQ GTKVEIK] NV1 NV07 2001 Amino Acid Sequence:  (SEQ ID NO: 1320) YTFGCPDRYCDRAATGGGSSGGSISSGLLSGRSDNHGGSEIVLTQS PATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNR ATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQG TKVEIK [Spacer (SEQ ID NO: 918)] [NV1 NV07 2001 (SEQ ID NO: 1322)]  Nucleotide Sequence:  (SEQ ID NO: 1323) [CAAGGCCAGTCTGGCCAAGGT][TATACGTTTGGTTGCCCTGATAGG TATTGCGATCGTGCGGCGACGGGAGGTGGCTCGAGCGGCGGCTCTATCT CTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGAT CGTGCTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAG CCACCCTGAGCTGTAGAGCCAGCCAGAGCGTGTCCAGCTACCTGGCCTGG TATCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTACGATGCCAG CAATAGAGCCACCGGCATCCCCGCCAGATTTTCCGGCTCTGGCAGCGGCA CCGACTTCACCCTGACCATCAGCTCCCTGGAACCCGAGGACTTCGCCGTG TACTACTGCCAGCAGAGCAGCAACTGGCCCCGGACATTTGGCCAGGGCAC CAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [NV1 NV08 2001 (SEQ ID NO: 1324) Amino Acid Sequence:  (SEQ ID NO: 1325) [QGQSGQG][RGCPDFNPPSHCYTAGGGSSGGSISSGLLSGRSDNHGGSE IVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDA SNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQG TKVEIK] NV1 NV08 2001 Amino Acid Sequence:  (SEQ ID NO: 1324) RGCPDFNPPSHCYTAGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPA TLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK [Spacer (SEQ ID NO: 918)] [NV1 NV08 2001 (SEQ ID NO: 1326)]  Nucleotide Sequence:  (SEQ ID NO: 1327) [CAAGGCCAGTCTGGCCAAGGT][CGTGGTTGTCCGGATTTTAATCCTCC TTCTCATTGCTATACTGCTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGATCGT GCTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAGCC ACCCTGAGCTGTAGAGCCAGCCAGAGCGTGTCCAGCTACCTGGCCTGGTA TCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTACGATGCCAGC AATAGAGCCACCGGCATCCCCGCCAGATTTTCCGGCTCTGGCAGCGGCAC CGACTTCACCCTGACCATCAGCTCCCTGGAACCCGAGGACTTCGCCGTGT ACTACTGCCAGCAGAGCAGCAACTGGCCCCGGACATTTGGCCAGGGCAC CAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [NV1 NV09 2001 (SEQ ID NO: 1328) Amino Acid Sequence:  (SEQ ID NO: 1329) [QGQSGQG][RDYCGPQSPDYCHEIGGGSSGGSISSGLLSGRSDNHGG SEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFG QGTKVEIK] NV1 NV09 2001 Amino Acid Sequence:  (SEQ ID NO: 1328) RDYCGPQSPDYCHEIGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPA TLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK [Spacer (SEQ ID NO: 918)] [NV1 NV09 2001 (SEQ ID NO: 1330)]  Nucleotide Sequence:  (SEQ ID NO: 1331) [CAAGGCCAGTCTGGCCAAGGT][AGGGATTATTGCGGGCCTCAGAGTCC TGATTATTGCCATGAGATTGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGATCGT GCTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAGCC ACCCTGAGCTGTAGAGCCAGCCAGAGCGTGTCCAGCTACCTGGCCTGGTA TCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTACGATGCCAGCA ATAGAGCCACCGGCATCCCCGCCAGATTTTCCGGCTCTGGCAGCGGCACC GACTTCACCCTGACCATCAGCTCCCTGGAACCCGAGGACTTCGCCGTGTA CTACTGCCAGCAGAGCAGCAACTGGCCCCGGACATTTGGCCAGGGCACCA AGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [NV1 NV10 2001 (SEQ ID NO: 1332) Amino Acid Sequence:  (SEQ ID NO: 1333) [QGQSGQG][PNKPCPDLQCYVTNYGGGSSGGSISSGLLSGRSDNHGG SEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFG QGTKVEIK] NV1 NV10 2001 Amino Acid Sequence:  (SEQ ID NO: 1332) PNKPCPDLQCYVTNYGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPA TLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK [Spacer (SEQ ID NO: 918)] [NV1 NV10 2001 (SEQ ID NO: 1334)]  Nucleotide Sequence:  (SEQ ID NO: 1335) [CAAGGCCAGTCTGGCCAAGGT][CCGAATAAGCCTTGCCCGGATCTGC AGTGCTATGTGACGAATTATGGAGGTGGCTCGAGCGGCGGCTCTATCTCT TCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGATCG TGCTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAGCC ACCCTGAGCTGTAGAGCCAGCCAGAGCGTGTCCAGCTACCTGGCCTGGTA TCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTACGATGCCAGC AATAGAGCCACCGGCATCCCCGCCAGATTTTCCGGCTCTGGCAGCGGCAC CGACTTCACCCTGACCATCAGCTCCCTGGAACCCGAGGACTTCGCCGTGT ACTACTGCCAGCAGAGCAGCAACTGGCCCCGGACATTTGGCCAGGGCACC AAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [NV1 NV11 2001 (SEQ ID NO: 1336) Amino Acid Sequence:  (SEQ ID NO: 1337) [QGQSGQG][PRVACGEPDLCYSNTGGGSSGGSISSGLLSGRSDNHGG SEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTF GQGTKVEIK] NV1 NV11 2001 Amino Acid Sequence:  (SEQ ID NO: 1336) PRVACGEPDLCYSNTGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPA TLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK [Spacer (SEQ ID NO: 918)] [NV1 NV11 2001 (SEQ ID NO: 1338)]  Nucleotide Sequence:  (SEQ ID NO: 1339) [CAAGGCCAGTCTGGCCAAGGT][CCGCGGGTTGCTTGCGGTGAGCCT GATCTTTGCTATTCTAATACTGGAGGTGGCTCGAGCGGCGGCTCTATCTC TTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGATC GTGCTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAGC CACCCTGAGCTGTAGAGCCAGCCAGAGCGTGTCCAGCTACCTGGCCTGGT ATCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTACGATGCCAGC AATAGAGCCACCGGCATCCCCGCCAGATTTTCCGGCTCTGGCAGCGGCAC CGACTTCACCCTGACCATCAGCTCCCTGGAACCCGAGGACTTCGCCGTGT ACTACTGCCAGCAGAGCAGCAACTGGCCCCGGACATTTGGCCAGGGCAC CAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [NV1 NV12 2001 (SEQ ID NO: 1340) Amino Acid Sequence:  (SEQ ID NO: 1341) [QGQSGQG][RGCKKHTISTLTCPDGGGSSGGSISSGLLSGRSDNHGG SEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFG QGTKVEIK] NV1 NV12 2001 Amino Acid Sequence:  (SEQ ID NO: 1340) RGCKKHTISTLTCPDGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK [Spacer (SEQ ID NO: 918)] [NV1 NV12 2001 (SEQ ID NO: 1342)]  Nucleotide Sequence:  (SEQ ID NO: 1343) [CAAGGCCAGTCTGGCCAAGGT][CGGGGGTGCAAGAAGCATACTATT TCGACGCTTACGTGCCCTGATGGAGGTGGCTCGAGCGGCGGCTCTA TCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAG ATCGTGCTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAG AGCCACCCTGAGCTGTAGAGCCAGCCAGAGCGTGTCCAGCTACCTGGCCT GGTATCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTACGATGCC AGCAATAGAGCCACCGGCATCCCCGCCAGATTTTCCGGCTCTGGCAGCGG CACCGACTTCACCCTGACCATCAGCTCCCTGGAACCCGAGGACTTCGCCG TGTACTACTGCCAGCAGAGCAGCAACTGGCCCCGGACATTTGGCCAGGGC ACCAAGGTGGAAATCAAG] Human Kappa Constant  (SEQ ID NO: 1344) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC Human Kappa Constant  (SEQ ID NO: 1345) CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATC CCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGT AACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAG CCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAG AGCTTCAACAGGGGAGAGTGT NV1 HC Variable  (SEQ ID NO: 1346) QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAV IWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATND DYWGQGTLVTVSS NV1 HC Variable  (SEQ ID NO: 1347) CAGGTGCAGCTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCAGAAG CCTGAGACTGGACTGCAAGGCCAGCGGCATCACCTTCAGCAACAGCGGCA TGCACTGGGTGCGCCAGGCCCCTGGAAAAGGCCTGGAATGGGTGGCCGTG ATTTGGTACGACGGCAGCAAGCGGTACTACGCCGACAGCGTGAAGGGCCG GTTCACCATCAGCCGGGACAACAGCAAGAATACCCTGTTCCTGCAGATGA ACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCACCAACGAC GACTATTGGGGCCAGGGCACACTCGTGACCGTGTCCTCT hIgG4 S228P Constant  (SEQ ID NO: 1348) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK hIgG4 S228P Constant  (SEQ ID NO: 1349) GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTGTAGCAGAAG CACCAGCGAGTCTACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCC CCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTG CACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCAAGACCTACACCTGTA ACGTGGACCACAAGCCCAGCAACACCAAGGTGGACAAGCGGGTGGAATCT AAGTACGGCCCTCCCTGCCCTCCTTGCCCAGCCCCTGAATTTCTGGGGGG ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT CCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGAAGAC CCTGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC CAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG TGCAAGGTCTCCAACAAAGGCCTGCCCAGCTCCATCGAGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAGACTCACCGTGGACAAGAGCAGGTGGCAGGAAGGG AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC GCAGAAGAGCCTCTCCCTGTCTCTGGGTAAA

Example 21: Characterization of Activatable Anti-PD1 NV1 Antibodies

This example describes activatable anti-PD1 NV1 antibodies with reduced binding to hPD1.

Masking efficiencies were evaluated by standard plate ELISA. Briefly, human PD1-Fc (R and D systems, Minneapolis, Minn.) was adsorbed to the wells of a 96-well ELISA plate. Purified NV1 and activatable NV1 antibodies were applied to the plate in serial dilution and allowed to bind. Bound antibody and activatable antibodies were detected with anti-hu Fab-HRP conjugate (Sigma, St Louis, Mo.) and visualized with the chromogenic substrate TMB (Thermo Scientific, Rockford, Ill.). Plots were generated in Prizm (Sigma Plot). All activatable NV1 antibodies showed decreased binding compared with the parental NV1, as shown in FIG. 26.

Example 22: Activatable Anti-PD1 Pembrolizumab Masking Moieties

This example describes identification of masking moieties (MM) that reduce binding of anti-PD1 pembrolizumab antibody to its target.

Anti-PD1 Pembrolizumab (PM1) (see, e.g., U.S. Pat. No. 8,354,509) was used to screen libraries using a method similar to that described in PCT International Publication Number WO 2010/081173, published 15 Jul. 2010. The screening consisted of one round of MACS and three rounds of FACS sorting. For the initial MACS, approximately 1.6×10¹² cells were incubated with PM1 antibody at a concentration of 200 nM, and approximately 8×10⁶ binders were collected using Protein-A Dynabeads (Life Technologies, Carlsbad, Calif.). FACS rounds were conducted labeling cells with DyLight 650 (Thermo-Fisher) labeled PM1 antibody and collecting cells with strongest fluorescence as follows: 100 nM PM1-650 collecting brightest 10% for FACS round 1 (5.6×10⁵ for F1), 10 nM PM1-650 collecting brightest 1.5% for FACS round 2 (1.4×10⁴ for F2), 1 nM PM1-650 collecting all binders above background (4,000) and the top 0.2% (820) for FACS round 3 (F3). Individual peptide clones from the two F3 populations were identified by sequence analysis (Table 20), and selected peptide clones PM01-PM12 were subsequently verified for their ability to bind PM1 (masking moiety PM001 is also referred to herein as PM01 and/or PM-01; masking moiety PM002 is also referred to herein as PM02 and/or PM-02, and so on).

TABLE 20 Mask Amino Acid Sequence SEQ ID NO: PM01 GCDFTSAKHNCGSGW 1351 PM02 VGSNCWTGPACALTS 1352 PM03 FCAVMFDFLSDRCLH 1353 PM04 FCPPWLDYLGNKCMT 1354 PM05 MSCWDFSSAQGCGQH 1355 PM06 LMCADLHYNHYNCKY 1356 PM07 ELCGWQSFSGVCTSE 1357 PM08 WTYENCWASCQPHLE 1358 PM09 KLTEDFSSAA 1359 PM10 VGQSCFSGLVCDRQL 1360 PM11 ISHYCFSGKSCRD 1361 PM12 HCIPDFTSAAGDCMR 1362 PM13 RLVSAYSFS 1363 PM14 KFHHSHPLVHDFTSA 1364 PM15 ASYPDFSSANGVGLR 1365 PM16 GLATTLSNVDFTSAG 1366 PM17 DFTSANSAFSGDAST 1367 PM18 GRLPGHSVVDFTSAW 1368 PM19 SGSFYSSSAFDFTSA 1369 PM20 CDDFTSAQHSRINEC 1370 PM21 CDFTSAQGKKCRTAL 1371 PM22 YYIDKYQSPSYGPVL 1372 PM23 FSVARARSSADFTSS 1373 PM24 DSDFTSAGSADSRSR 1374 PM25 CDFTSATSISKRCDH 1375 PM26 IESSASSWGLQASRN 1376 PM27 PRYHNLNFTTPALSPGS 1377 PM28 DLFARFPLDRDFTSA 1378 PM29 HCNFTTPPYCSSTLW 1379 PM30 NVPIILLTDRQLLSG 1380 PM31 NPTACDFTSSMATYC 1381 PM32 FVRTVRFSNSMFSVP 1382 PM33 YDFSSASNSSPSRQT 1383 PM34 AHPDFSSAMRGNLLG 1384 PM35 SSHVVHKDFTSANSR 1385 PM36 CPDFTSANGGGCWQM 1386 PM37 SLGQSYPTDFTCPGC 1387 PM38 ASMRSHEQRDFTSAY 1388 PM39 SCQFWFTLCSGGVFH 1389 PM40 PYPNNRTGMHDFTSA 1390 PM41 KPFPIDFTSAGTSGT 1391 PM42 SIKSFIPRDDFTSAA 1392 PM43 GIKNPATPFVDFTSA 1393 PM44 LSHTYPRGSSTIEAS 1394 PM45 PSLDFSSAT 1395 PM46 AFTPRIAPTFDVMKE 1396 PM47 LCGLQIPPDCERS 1397 PM48 AAKMVSHSERDFTSA 1398 PM49 VSVECFSGMQCPHYY 1399 PM50 ASKCRLPCMASTQIY 1400 PM51 GLRSCNIYFSIPCTY 1401 PM52 RGTSDGTLDFTTARS 1402 PM53 SMYPSASRLLHPQYP 1403 PM54 HCISCYDFTSAAGSF 1404 PM55 SSGRWGDAWACARIC 1405 PM56 RVFSDFTSASHSFGG 1406 PM57 TDRHSASGRDFTSAH 1407 PM58 AHCEDFSSAERIATMGC 1408 PM59 ACDPYSFSIPCDDRL 1409 PM60 NSPFTLSHIYDR 1410 PM61 IGTNFTTPSAFVAFP 1411 PM62 RDAFPIYRNADFSTP 1412 PM63 SIPNASSYNFTSSSG 1413 PM64 AGIPDKRHTYDFTSA 1414 PM65 WPLAHDSRDWNFTTP 1415 PM66 RHSPSSGHVDFTSAG 1416 PM67 SCFAWTDPVWNRCSW 1417 PM68 MPCDWTGPGKIWCGG 1418 PM69 RDCDFSTANFRSCNK 1419 PM70 LSCVVSPNYLHCNDH 1420 PM71 FVCGLYSFSGVCQGV 1421 PM72 IGLMCFSGLQCPMLA 1422 PM73 PGMNCFSGEICQMST 1423 PM74 GDVGSCWASCGLQGG 1424 PM75 SQFQDCWASCGASFT 1425 PM76 VGSLNCWYSCGDIWL 1426 PM77 MCESWLNFLGDQCGM 1427 PM78 RCMISQSSFSGMCGM 1428 PM79 NCAPWTSNMSNHCLK 1429 PM80 LCGVGSATGLELCGV 1430 PM81 GCDFSSLGGRQPCTP 1431 PM82 MGCNFTTYPYHTCNT 1432 PM83 GSCDFTSGAGKKCGS 1433 PM84 VSCDFTSSHARMCSR 1434 PM85 MRCTDFYYNHTNCIG 1435 PM86 RSCDFTSAANKYCAT 1436 PM87 LYCDSFSVPRPNCAP 1437 PM88 NSCDFTSARVSKCST 1438 PM89 STCSDNFTTPMPCNT 1439 PM90 DICNDRPNLTHCHYF 1440 PM91 LRCDDFTSAIGCRGY 1441 PM92 EGCDFTSALHSCNNY 1442 PM93 RKGCGDFTSASCFVV 1443 PM94 GMLCAGSSFGLCESM 1444 PM95 RESCFGSSLGLCTNK 1445 PM96 ILRCYDIPTNCMNFN 1446 PM97 NSECTFGAMYCRNKP 1447 PM98 ASGCFDEDIRCSGGA 1448 PM99 HYFCNQSNPSCQTAP 1449 PM100 AMGCELGGAGCIGSP 1450 PM101 TLKCHMPRKLCANDP 1451 PM102 RPACRDLPHNCITST 1452 PM103 QMSCHGNFTTCHSNP 1453 PM104 LTGCARGARPCRLRV 1454 PM105 WSELCLAGPSCGWVG 1455 PM106 VTKSCWQLPHCITAP 1456 PM107 KAASCPHNQICNMTA 1457 PM108 VSKNCFSGMMCPVFA 1458 PM109 NRSSCWTGPTCHVLH 1459 PM110 ARTGCSGPVCLNDVS 1460 PM111 STRTCLAFTCINGNT 1461 PM112 MLDGNCWYACSYKNT 1462 PM113 FSRSDCWSACAPWRV 1463 PM114 GGRMDCWASCQPLSR 1464 PM115 NSPHSCMTNCDFTSA 1465

Example 23: Activatable Anti-PD1 Pembrolizumab Antibodies

This example describes examples of activatable anti-PD1 Pembrolizumab antibodies of the disclosure.

Activatable anti-PD1 PM1 antibodies comprising an anti-PD1 PM1 masking moiety, a cleavable moiety, and an anti-PD1 PM1 antibody of the disclosure were produced according to methods similar to those described in PCT Publication Nos. WO 2009/025846 and WO 2010/081173. One embodiment of the disclosure is an anti-PD-1 activatable antibody comprising a heavy chain comprising amino acid sequence SEQ ID NO: 1514. One embodiment of the disclosure is an anti-PD-1 activatable antibody comprising a light chain comprising amino acid sequence SEQ ID NO: 638. One embodiment of the disclosure is an anti-PD-1 activatable antibody comprising a heavy chain comprising amino acid sequence SEQ ID NO: 1514 and a light chain comprising amino acid sequence SEQ ID NO: 638. One embodiment of the disclosure is an anti-PD-1 activatable antibody comprising the CDRs of an anti-PD-1 activatable antibody comprising a heavy chain comprising amino acid sequence SEQ ID NO: 1514 and a light chain comprising amino acid sequence SEQ ID NO: 638.

The amino acid and nucleic acid sequences of several activatable anti-PD1 antibody variable domains of the disclosure are provided below. Antibodies were produced as hIgG4 containing a single amino acid substitution, S228P (Angal, et al. 1993. Mol Immunol 30:105-8.) HC and hK LC format.

PM1 Light Chain Amino Acid Sequence:  (SEQ ID NO: 638) EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRL LIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPL TFGGGTKVEIK [Spacer (SEQ ID NO: 362)] [PM1 PM01 2001 (SEQ ID NO: 1466)]   Amino Acid Sequence: (SEQ ID NO: 1467) [QGQSGQG][CDFTSAKHNCGSGWGSSGGSISSGLLSGRSDNHGGSEI VLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLI YLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTF GGGTKVEIK] PM1 PM01 2001 Amino Acid Sequence:  (SEQ ID NO: 1466) CDFTSAKHNCGSGWGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATLSLS PGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK [Spacer (SEQ ID NO: 1126)] [PM1 PM01 2001 (SEQ ID NO: 1468)]  Nucleotide Sequence:  (SEQ ID NO: 1469) [CAAGGCCAGTCTGGCCAAGGA][TGCGATTTTACTTCTGCCAAGCAC AATTGCGGCTCTGGCTGGGGCTCGAGCGGCGGCTCTATCTCTTCCGGACT GCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGATCGTGCTGACAC AGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAGCCACCCTGAGC TGTAGAGCCTCTAAGGGCGTGTCCACCAGCGGCTACAGCTACCTGCACTG GTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTACCTGGCCT CCTACCTGGAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGGCAGCGGC ACCGACTTCACCCTGACAATCAGCAGCCTGGAACCCGAGGACTTCGCCGT GTACTACTGCCAGCACAGCAGGGACCTGCCCCTGACATTTGGCGGAGGCA CCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PM1 PM02 2001 (SEQ ID NO: 1470)]  Amino Acid Sequence:  (SEQ ID NO: 1471) [QGQSGQG][VGSNCWTGPACALTSGGGSSGGSISSGLLSGRSDNHGG SEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPR LLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLP LTFGGGTKVEIK] PM1 PM02 2001 Amino Acid Sequence:  (SEQ ID NO: 1470) VGSNCWTGPACALTSGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESG VPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK [Spacer (SEQ ID NO: 918)] [PM1 PM02 2001 (SEQ ID NO: 1472)]  Nucleotide Sequence:  (SEQ ID NO: 1473) [CAAGGCCAGTCTGGCCAAGGT][GTTGGGTCGAATTGCTGGACGGGGCC GGCGTGCGCTTTGACGTCGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGATCGTG CTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAGCCAC CCTGAGCTGTAGAGCCTCTAAGGGCGTGTCCACCAGCGGCTACAGCTACC TGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTAC CTGGCCTCCTACCTGGAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGG CAGCGGCACCGACTTCACCCTGACAATCAGCAGCCTGGAACCCGAGGACT TCGCCGTGTACTACTGCCAGCACAGCAGGGACCTGCCCCTGACATTTGGC GGAGGCACCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PM1 PM03 2001 (SEQ ID NO: 1474)]  Amino Acid Sequence:  (SEQ ID NO: 1475) [QGQSGQG][FCAVMFDFLSDRCLHGGGSSGGSISSGLLSGRSDNHGGSE IVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLL IYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLT FGGGTKVEIK] PM1 PM03 2001 Amino Acid Sequence:  (SEQ ID NO: 1474) FCAVMFDFLSDRCLHGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESG VPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK [Spacer (SEQ ID NO: 918)] [PM1 PM03 2001 (SEQ ID NO: 1476)]  Nucleotide Sequence:  (SEQ ID NO: 1477) [CAAGGCCAGTCTGGCCAAGGT][TTTTGCGCTGTGATGTTTGATTTTCT GTCTGATCGGTGCCTGCATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGATCGTG CTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAGCCAC CCTGAGCTGTAGAGCCTCTAAGGGCGTGTCCACCAGCGGCTACAGCTACC TGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTAC CTGGCCTCCTACCTGGAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGG CAGCGGCACCGACTTCACCCTGACAATCAGCAGCCTGGAACCCGAGGACT TCGCCGTGTACTACTGCCAGCACAGCAGGGACCTGCCCCTGACATTTGGC GGAGGCACCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PM1 PM04 2001 (SEQ ID NO: 1478)]  Amino Acid Sequence:  (SEQ ID NO: 1479) [QGQSGQG][FCPPWLDYLGNKCMTGGGSSGGSISSGLLSGRSDNHGGSE IVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLL IYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLT FGGGTKVEIK] PM1 PM04 2001 Amino Acid Sequence:  (SEQ ID NO: 1478) FCPPWLDYLGNKCMTGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESG VPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK [Spacer (SEQ ID NO: 918)] [PM1 PM04 2001 (SEQ ID NO: 1480)]  Nucleotide Sequence:  (SEQ ID NO: 1481) [CAAGGCCAGTCTGGCCAAGGT][TTTTGCCCGCCGTGGTTGGATTATTT GGGTAATAAGTGCATGACGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGATCGTG CTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAGCCAC CCTGAGCTGTAGAGCCTCTAAGGGCGTGTCCACCAGCGGCTACAGCTACC TGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTAC CTGGCCTCCTACCTGGAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGG CAGCGGCACCGACTTCACCCTGACAATCAGCAGCCTGGAACCCGAGGACT TCGCCGTGTACTACTGCCAGCACAGCAGGGACCTGCCCCTGACATTTGGC GGAGGCACCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PM1 PM05 2001 (SEQ ID NO: 1482)]  Amino Acid Sequence:  (SEQ ID NO: 1483) [QGQSGQG][MSCWDFSSAQGCGQHGGGSSGGSISSGLLSGRSDNHGGSE IVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLL IYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLT FGGGTKVEIK] PM1 PM05 2001 Amino Acid Sequence:  (SEQ ID NO: 1482) MSCWDFSSAQGCGQHGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESG VPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK [Spacer (SEQ ID NO: 918)] [PM1 PM05 2001 (SEQ ID NO: 1484)]  Nucleotide Sequence:  (SEQ ID NO: 1485) [CAAGGCCAGTCTGGCCAAGGT][ATGTCTTGCTGGGATTTTTCTTCGGC TCAGGGGTGCGGTCAGCATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGATCGTG CTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAGCCAC CCTGAGCTGTAGAGCCTCTAAGGGCGTGTCCACCAGCGGCTACAGCTACC TGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTAC CTGGCCTCCTACCTGGAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGG CAGCGGCACCGACTTCACCCTGACAATCAGCAGCCTGGAACCCGAGGACT TCGCCGTGTACTACTGCCAGCACAGCAGGGACCTGCCCCTGACATTTGGC GGAGGCACCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PM1 PM06 2001 (SEQ ID NO: 1486)]  Amino Acid Sequence:  (SEQ ID NO: 1487) [QGQSGQG][LMCADLHYNHYNCKYGGGSSGGSISSGLLSGRSDNHGGSE IVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLL IYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLT FGGGTKVEIK] PM1 PM06 2001 Amino Acid Sequence:  (SEQ ID NO: 1486) LMCADLHYNHYNCKYGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESG VPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK [Spacer (SEQ ID NO: 918)] [PM1 PM06 2001 (SEQ ID NO: 1488)]  Nucleotide Sequence:  (SEQ ID NO: 1489) [CAAGGCCAGTCTGGCCAAGGT][TTGATGTGCGCTGATTTGCATTATAA TCATTATAATTGCAAGTATGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGATCGTG CTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAGCCAC CCTGAGCTGTAGAGCCTCTAAGGGCGTGTCCACCAGCGGCTACAGCTACC TGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTAC CTGGCCTCCTACCTGGAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGG CAGCGGCACCGACTTCACCCTGACAATCAGCAGCCTGGAACCCGAGGACT TCGCCGTGTACTACTGCCAGCACAGCAGGGACCTGCCCCTGACATTTGGC GGAGGCACCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PM1 PM07 2001 (SEQ ID NO: 1490)]  Amino Acid Sequence:  (SEQ ID NO: 1491) [QGQSGQG][ELCGWQSFSGVCTSEGGGSSGGSISSGLLSGRSDNHGGSE IVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLL IYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLT FGGGTKVEIK] PM1 PM07 2001 Amino Acid Sequence:  (SEQ ID NO: 1490) ELCGWQSFSGVCTSEGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESG VPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK [Spacer (SEQ ID NO: 918)] [PM1 PM07 2001 (SEQ ID NO: 1492)]  Nucleotide Sequence:  (SEQ ID NO: 1493) [CAAGGCCAGTCTGGCCAAGGT][GAGTTGTGCGGTTGGCAGAGTTTTTC GGGGGTTTGCACTAGTGAGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGATCGTG CTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAGCCAC CCTGAGCTGTAGAGCCTCTAAGGGCGTGTCCACCAGCGGCTACAGCTACC TGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTAC CTGGCCTCCTACCTGGAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGG CAGCGGCACCGACTTCACCCTGACAATCAGCAGCCTGGAACCCGAGGACT TCGCCGTGTACTACTGCCAGCACAGCAGGGACCTGCCCCTGACATTTGGC GGAGGCACCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 362)] [PM1 PM08 2001 (SEQ ID NO: 1494)]  Amino Acid Sequence:  (SEQ ID NO: 1495) [QGQSGQG][WTYENCWASCQPHLEGGGSSGGSISSGLLSGRSDNHGGSE IVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLL IYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLT FGGGTKVEIK] PM1 PM08 2001 Amino Acid Sequence:  (SEQ ID NO: 1494) WTYENCWASCQPHLEGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESG VPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK [Spacer (SEQ ID NO: 918)] [PM1 PM08 2001 (SEQ ID NO: 1496)]  Nucleotide Sequence:  (SEQ ID NO: 1497) [CAAGGCCAGTCTGGCCAAGGT][TGGACTTATGAGAATTGCTGGGCTTC GTGCCAGCCTCATTTGGAGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGATCGTG CTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAGCCAC CCTGAGCTGTAGAGCCTCTAAGGGCGTGTCCACCAGCGGCTACAGCTACC TGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTAC CTGGCCTCCTACCTGGAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGG CAGCGGCACCGACTTCACCCTGACAATCAGCAGCCTGGAACCCGAGGACT TCGCCGTGTACTACTGCCAGCACAGCAGGGACCTGCCCCTGACATTTGGC GGAGGCACCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 913)] [PM1 PM09 2001 (SEQ ID NO: 1498)]  Amino Acid Sequence:  (SEQ ID NO: 1499) [QGQSGQ][KLTEDFSSAAGSSGGSISSGLLSGRSDNHGGSEIVLTQSPA TLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLE SGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVE IK] PM1 PM09 2001 Amino Acid Sequence:  (SEQ ID NO: 1498) KLTEDFSSAAGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATLSLSPGER ATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSG SGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK [Spacer (SEQ ID NO: 1518)] [PM1 PM09 2001 (SEQ ID NO: 1500)]  Nucleotide Sequence:  (SEQ ID NO: 1501) [CAAGGCCAGTCTGGCCAA][AAGCTTACTGAGGATTTTTCTAGCGCAGC AGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCG ACAATCACGGCGGATCCGAGATCGTGCTGACACAGAGCCCTGCCACCCTG TCTCTGAGCCCTGGCGAAAGAGCCACCCTGAGCTGTAGAGCCTCTAAGGG CGTGTCCACCAGCGGCTACAGCTACCTGCACTGGTATCAGCAGAAGCCCG GCCAGGCCCCCAGACTGCTGATCTACCTGGCCTCCTACCTGGAAAGCGGC GTGCCCGCCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGAC AATCAGCAGCCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCACA GCAGGGACCTGCCCCTGACATTTGGCGGAGGCACCAAGGTGGAAATC AAG] [Spacer (SEQ ID NO: 362)] [PM1 PM10 2001 (SEQ ID NO: 1502)]  Amino Acid Sequence:  (SEQ ID NO: 1503) [QGQSGQG][VGQSCFSGLVCDRQLGGGSSGGSISSGLLSGRSDNHGGSE IVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLL IYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLT FGGGTKVEIK] PM1 PM10 2001 Amino Acid Sequence:  (SEQ ID NO: 1502) VGQSCFSGLVCDRQLGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESG VPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK [Spacer (SEQ ID NO: 918)] [PM1 PM10 2001 (SEQ ID NO: 1504)]  Nucleotide Sequence:  (SEQ ID NO: 1505) [CAAGGCCAGTCTGGCCAAGGT][GTGGGGCAGAGTTGCTTTTCTGGGCT GGTTTGCGATAGGCAGCTGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGATCGTG CTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAGCCAC CCTGAGCTGTAGAGCCTCTAAGGGCGTGTCCACCAGCGGCTACAGCTACC TGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTAC CTGGCCTCCTACCTGGAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGG CAGCGGCACCGACTTCACCCTGACAATCAGCAGCCTGGAACCCGAGGACT TCGCCGTGTACTACTGCCAGCACAGCAGGGACCTGCCCCTGACATTTGGC GGAGGCACCAAGGTGGAAATCAAG] [Spacer (SEQ ID NO: 913)] [PM1 PM11 2001 (SEQ ID NO: 1506)]  Amino Acid Sequence:  (SEQ ID NO: 1507) [QGQSGQ][ISHYCFSGKSCRDGSSGGSISSGLLSGRSDNHGGSEIVLTQ SPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLAS YLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGT KVEIK] PM1 PM11 2001 Amino Acid Sequence:  (SEQ ID NO: 1506) ISHYCFSGKSCRDGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATLSLSP GERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPAR FSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK [Spacer (SEQ ID NO: 1518)] [PM1 PM11 2001 (SEQ ID NO: 1508)]  Nucleotide Sequence:  (SEQ ID NO: 1509) [CAAGGCCAGTCTGGCCAA][ATCTCTCACTATTGTTTCAGTGGCAAATC CTGCAGGGACGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCG GCAGATCCGACAATCACGGCGGATCCGAGATCGTGCTGACACAGAGCCCT GCCACCCTGTCTCTGAGCCCTGGCGAAAGAGCCACCCTGAGCTGTAGAGC CTCTAAGGGCGTGTCCACCAGCGGCTACAGCTACCTGCACTGGTATCAGC AGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTACCTGGCCTCCTACCTG GAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGGCAGCGGCACCGACTT CACCCTGACAATCAGCAGCCTGGAACCCGAGGACTTCGCCGTGTACTACT GCCAGCACAGCAGGGACCTGCCCCTGACATTTGGCGGAGGCACCAAGGTG GAAATCAAG] [Spacer (SEQ ID NO: 362)] [PM1 PM12 2001 (SEQ ID NO: 1510)]  Amino Acid Sequence:  (SEQ ID NO: 1511) [QGQSGQG][HCIPDFTSAAGDCMRGGGSSGGSISSGLLSGRSDNHGGSE IVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLL IYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLT FGGGTKVEIK] PM1 PM12 2001 Amino Acid Sequence:  (SEQ ID NO: 1510) HCIPDFTSAAGDCMRGGGSSGGSISSGLLSGRSDNHGGSEIVLTQSPATL SLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESG VPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK [Spacer (SEQ ID NO: 918)] [PM1 PM12 2001 (SEQ ID NO: 1512)]  Nucleotide Sequence:  (SEQ ID NO: 1513) [CAAGGCCAGTCTGGCCAAGGT][CATTGCATTCCTGATTTTACTTCTGC TGCTGGTGATTGCATGAGGGGAGGTGGCTCGAGCGGCGGCTCTATCTCTT CCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGATCCGAGATCGTG CTGACACAGAGCCCTGCCACCCTGTCTCTGAGCCCTGGCGAAAGAGCCAC CCTGAGCTGTAGAGCCTCTAAGGGCGTGTCCACCAGCGGCTACAGCTACC TGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTAC CTGGCCTCCTACCTGGAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGG CAGCGGCACCGACTTCACCCTGACAATCAGCAGCCTGGAACCCGAGGACT TCGCCGTGTACTACTGCCAGCACAGCAGGGACCTGCCCCTGACATTTGGC GGAGGCACCAAGGTGGAAATCAAG] PM1 HC Variable  (SEQ ID NO: 1514) QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGG INPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRD YRFDMGFDYWGQGTTVTVSS PM1 HC Variable  (SEQ ID NO: 1515) CAGGTGCAGCTGGTGCAGTCTGGCGTGGAAGTGAAGAAACCAGGCGCCAG CGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTACCAACTACTACA TGTACTGGGTGCGCCAGGCCCCAGGCCAGGGACTGGAATGGATGGGCGGC ATCAACCCCAGCAACGGCGGCACCAACTTCAACGAGAAGTTCAAGAACAG AGTGACCCTGACCACCGACAGCAGCACCACCACCGCCTACATGGAACTGA AGTCCCTGCAGTTCGACGACACCGCCGTGTACTACTGCGCCAGACGGGAC TACAGATTCGACATGGGCTTCGACTACTGGGGCCAGGGCACAACCGTGAC CGTGTCTAGT

Example 24: Characterization of Activatable Anti-PD1 PM1 Antibodies

This example describes activatable anti-PD1 PM1 antibodies with reduced binding to hPD1.

Masking efficiencies were evaluated by standard plate ELISA. Briefly, human PD1-Fc (R and D systems, Minneapolis, Minn.) was adsorbed to the wells of a 96-well ELISA plate. Purified PM1 and activatable PM1 antibodies were applied to the plate in serial dilution and allowed to bind. Bound antibody and activatable antibodies were detected with anti-hu Fab-HRP conjugate (Sigma, St Louis, Mo.) and visualized with the chromogenic substrate TMB (Thermo Scientific, Rockford, Ill.). Plots were generated in Prizm (Sigma Plot). All activatable PM1 antibodies showed decreased binding compared with the parental PM1, as shown in FIGS. 27A, 27B, and 27C.

Example 25: Characterization of Anti-PD1 Activatable Antibodies of the Disclosure

The binding properties of several masking moieties of the disclosure identified for A1.5 (PD), NV1 (NV) and PM1 (PM) antibodies were evaluated. Eight peptide clones from each mask discovery effort were grown overnight at 37° C., 850 rpm in 2 mL deep well plates in LB+Chloramphenicol+0.2% glucose, diluted 1:20 in LB+Chloramphenicol and grown 105 minutes at 37° C., 850 rpm. Peptide expression was induced by the addition of 0.04% arabinose for 35 minutes, and clones were stained with 1 nM A1.5-DyLight 650, 1 nM PM1-Dylight 650 and 1 nM NV1-Dylight 650 in cold PBS+0.5% BSA. Clones were pelleted, the staining solution was removed, and cells were resuspended in PBS+1% formaldehyde. Fluorescence was measured on a MACSQuant Analyzer 10 flow cytometer manufactured by Miltenyi Biotec, Inc. (San Diego, Calif.). As shown in FIG. 28, masking moieties bound to the anti-PD1 antibody used in the library screening from which they were identified.

Example 26: Evaluation of Masking Moiety Binding by Effector Negative Activatable Antibodies

This example describes additional activatable anti-PD-1 J43 antibodies that exhibit reduced binding to mouse PD-1.

Examples of additional activatable antibodies of the disclosure comprising an IgG2a effector negative (EN) anti-PD1 antibody J43 (J43 m2a EN) and a variety of mask and substrate combinations were produced using techniques as described herein. The amino acid and nucleic acid sequences of the constant region of the IgG2a EN antibody are provided below. A plasmid encoding this effector negative Fc region, pFUSE-mIgG2Ae1-Fc, is available from InvivoGen, San Diego, Calif. The Fc region of mIgG2a was engineered by mutating the following amino acids to reduce binding to FcR and C1q: L235E and E318A/K320A/K322A.

+LT,1 >mIgG2a EN  (SEQ ID NO: 1516) AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGV HTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPR GPTIKPCPPCKCPAPNLEGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVS EDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGK AFACAVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTC MVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNW VERNSYSCSVVHEGLHNHHTTKSFSRTPGK >mIgG2a EN  (SEQ ID NO: 1517) GCCAAGACAACAGCCCCCAGCGTGTACCCTCTGGCCCCTGTGTGTGGCGA TACCACAGGCAGCTCTGTGACCCTGGGCTGCCTCGTGAAGGGCTACTTCC CTGAGCCAGTGACCCTGACCTGGAACAGCGGCTCTCTGTCTAGCGGCGTG CACACCTTTCCAGCCGTGCTGCAGAGCGACCTGTACACCCTGAGCAGCAG CGTGACCGTGACCAGCAGCACATGGCCCAGCCAGAGCATCACCTGTAACG TGGCCCACCCTGCCAGCTCCACCAAGGTGGACAAGAAGATCGAGCCCAGA GGCCCCACCATCAAGCCTTGCCCCCCTTGCAAATGCCCTGCCCCCAATCT GGAAGGCGGCCCTAGCGTGTTCATCTTCCCACCCAAGATCAAGGACGTGC TGATGATCAGCCTGAGCCCCATCGTGACCTGCGTGGTGGTGGACGTGTCC GAGGACGACCCCGATGTGCAGATCAGTTGGTTCGTGAACAACGTGGAAGT GCACACCGCCCAGACCCAGACACACAGAGAGGACTACAACAGCACCCTGA GAGTGGTGTCCGCCCTGCCCATCCAGCACCAGGATTGGATGAGCGGCAAG GCCTTCGCCTGCGCTGTGAACAACAAGGACCTGCCAGCCCCCATCGAGCG GACCATCTCTAAGCCTAAGGGCAGCGTGCGGGCTCCCCAGGTGTACGTGC TGCCTCCTCCAGAGGAAGAGATGACCAAGAAACAAGTGACACTGACATGC ATGGTCACCGACTTCATGCCCGAGGACATCTACGTGGAATGGACCAACAA CGGCAAGACCGAGCTGAACTACAAGAACACCGAGCCCGTGCTGGACAGCG ACGGCAGCTACTTCATGTACAGCAAGCTGCGGGTGGAAAAGAAAAACTGG GTGGAACGGAACAGCTACAGCTGCAGCGTGGTGCACGAGGGCCTGCACAA TCACCACACCACCAAGAGCTTCAGCCGGACCCCTGGAAAA

Masking efficiencies of several of these activatable antibodies were determined as described herein. The results are shown in FIG. 29A and FIG. 29B.

J43m2a EN Activatable Antibody LCs:

[Spacer (SEQ ID NO: 362)] [J43m2a EN MP8-2 2001 (SEQ ID NO: 1808)] Amino Acid Sequence: (SEQ ID NO: 1809) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDNHGGGS YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVLGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVT WKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHE GETVEKSLSPAECL] J43m2a EN MP8-2 2001 Amino Acid Sequence:  (SEQ ID NO: 1808) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDNHGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLG GPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVTWKANGATIND GVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSLSP AECL [Spacer (SEQ ID NO: 362)] [J43m2a EN MP8-2 2011 (SEQ ID NO: 1810)]  Amino Acid Sequence: (SEQ ID NO: 1811) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDNPGGGS YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVLGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVT WKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHE GETVEKSLSPAECL] J43m2a EN MP8-2 2011 Amino Acid Sequence:  (SEQ ID NO: 1810) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDNPGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLG GPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVTWKANGATIND GVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSLSP AECL [Spacer (SEQ ID NO: 362)] [J43m2a EN MP8-2 2012 (SEQ ID NO: 1812)]  Amino Acid Sequence: (SEQ ID NO: 1813) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSANPGGGS YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVLGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVT WKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHE GETVEKSLSPAECL] J43m2a EN MP8-2 2012 Amino Acid Sequence:  (SEQ ID NO: 1812) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSANPGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLG GPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVTWKANGATIND GVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSLSP AECL [Spacer (SEQ ID NO: 362)] [J43m2a EN MP8-2 2002 (SEQ ID NO: 1814)]  Amino Acid Sequence: (SEQ ID NO: 1815) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSGNHGGGS YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVLGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVT WKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHE GETVEKSLSPAECL] J43m2a EN MP8-2 2002 Amino Acid Sequence:  (SEQ ID NO: 1814) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSGNHGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLG GPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVTWKANGATIND GVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSLSP AECL [Spacer (SEQ ID NO: 362)] [J43m2a EN MP8-2 2003 (SEQ ID NO: 1816)]  Amino Acid Sequence: (SEQ ID NO: 1817) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSANPRGGG GSYELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYD DNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLY VFGSGTQLTVLGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSAT VTWKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVT HEGETVEKSLSPAECL] J43m2a EN MP8-2 2003 Amino Acid Sequence:  (SEQ ID NO: 1816) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSANPRGGGGSYELTQPPS ASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPE RISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTV LGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVTWKANGATI NDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSL SPAECL [Spacer (SEQ ID NO: 362)] [J43m2a EN MP8-2 2006 (SEQ ID NO: 1818)]  Amino Acid Sequence: (SEQ ID NO: 1819) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDDHGGGS YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVLGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVT WKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHE GETVEKSLSPAECL] J43m2a EN MP8-2 2006 Amino Acid Sequence:  (SEQ ID NO: 1818) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDDHGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLG GPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVTWKANGATIND GVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSLSP AECL [Spacer (SEQ ID NO: 362)] [J43m2a EN MP8-2 2007 (SEQ ID NO: 1820)]  Amino Acid Sequence: (SEQ ID NO: 1821) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDIHGGGS YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVLGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVT WKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHE GETVEKSLSPAECL] J43m2a EN MP8-2 2007 Amino Acid Sequence:  (SEQ ID NO: 1820) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDIHGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLG GPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVTWKANGATIND GVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSLSP AECL [Spacer (SEQ ID NO: 362)] [J43m2a EN MP8-2 2008 (SEQ ID NO: 1822)]  Amino Acid Sequence: (SEQ ID NO: 1823) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDQHGGGS YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVLGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVT WKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHE GETVEKSLSPAECL] J43m2a EN MP8-2 2008 Amino Acid Sequence:  (SEQ ID NO: 1822) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDQHGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLG GPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVTWKANGATIND GVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSLSP AECL [Spacer (SEQ ID NO: 362)] [J43m2a EN MP8-2 2009 (SEQ ID NO: 1824)]  Amino Acid Sequence: (SEQ ID NO: 1825) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDTHGGGS YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVLGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVT WKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHE GETVEKSLSPAECL] J43m2a EN MP8-2 2009 Amino Acid Sequence:  (SEQ ID NO: 1824) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDTHGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLG GPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVTWKANGATIND GVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSLSP AECL [Spacer (SEQ ID NO: 362)] [J43m2a EN MP8-2 2010 (SEQ ID NO: 1826)]  Amino Acid Sequence: (SEQ ID NO: 1827) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDYHGGGS YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVLGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVT WKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHE GETVEKSLSPAECL] J43m2a EN MP8-2 2010 Amino Acid Sequence:  (SEQ ID NO: 1826) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDYHGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLG GPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVTWKANGATIND GVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSLSP AECL [Spacer (SEQ ID NO: 362)] [J43m2a EN MP8-2 2013 (SEQ ID NO: 1828)]  Amino Acid Sequence: (SEQ ID NO: 1829) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSANIGGGS YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVLGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVT WKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHE GETVEKSLSPAECL] J43m2a EN MP8-2 2013 Amino Acid Sequence:  (SEQ ID NO: 1828) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSANIGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLG GPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVTWKANGATIND GVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSLSP AECL [Spacer (SEQ ID NO: 362)] [J43m2a EN MP8-2 2014 (SEQ ID NO: 1830)]  Amino Acid Sequence: (SEQ ID NO: 1831) [QGQSGQG][ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDNIGGGS YELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDN KRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVLGGPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVT WKANGATINDGVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHE GETVEKSLSPAECL] J43m2a EN MP8-2 2014 Amino Acid Sequence:  (SEQ ID NO: 1830) ACRICQDHPATKWNSGGGSSGGSISSGLLSGRSDNIGGGSYELTQPPSAS VNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERI SGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVFGSGTQLTVLG GPKSSPKVTVFPPSPEELRTNKATLVCLVNDFYPGSATVTWKANGATIND GVKTTKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSLSP AECL

Example 27: Activatable Anti-Mouse PD1 J43 m2a EN Antibodies Reduce Incidence of Diabetes in NOD Mice

In this Example, anti-PD1 J43 m2a EN (effector negative) activatable antibodies were analyzed for the ability to protect from PD1 m2a EN-mediated induction of diabetes in NOD mice.

The NOD mice, substrain NOD/ShiLtJ, were obtained from Jackson Laboratory at 8 weeks and acclimated on site for 2 weeks. At 10 weeks, mice were checked for diabetes prior to enrollment, grouped, and dosed as set forth in Table 21.

Dose Dose volume Group Count Gender Treatment (mg/kg) (mL/kg) Schedule Route 1 7 F mIgG2a (C1.18.4) 10 10 q7dx1 IP 2 7 F Anti-PD-1 J43 m2a EN 10 10 q7dx1 IP 3 7 F Anti-PD-1 J43 m2a EN 3 10 q7dx1 IP 4 7 F Anti-PD-1 J43 m2a EN 1 10 q7dx1 IP 5 7 F J43 MP7-1 2001 m2a EN 10 10 q7dx1 IP 6 7 F J43 MP7-1 2001 m2a EN 3 10 q7dx1 IP 7 7 F J43 MP8-2 2001 m2a EN 10 10 q7dx1 IP 8 7 F J43 MP8-2 2001 m2a EN 3 10 q7dx1 IP

FIG. 30 which plots % non-diabetic versus number of days post dose, shows that anti-PD-1 J43 antibody induced diabetes in NOD mice with increased frequency as dosage increased. At day eleven post dose, the percentage of non-diabetic mice for the antibody-treated groups was 29%, 43% and 71% for the 10 mg/kg, 3 mg/kg and 1 mg/kg dose groups, respectively. Activatable antibodies J43 MP7-1 2001 m2a EN and J43 MP8-2 2001 m2a EN required increased doses to induce diabetes at frequencies comparable to the parental antibody. At day eleven post dose with J43 MP7-1 2001 m2a EN, 71% of the 10 mg/kg group remained non-diabetic and 86% of the 3 mg/kg were non-diabetic. At day fourteen post dose with J43 MP8-2 2001 m2a EN, 86% of the 10 mg/kg group and 100% of the 3 mg/kg group remained non-diabetic.

Example 28. Activatable Anti-Mouse PD1 J43 m2a EN Antibodies Show Efficacy in the MC38 Syngeneic Tumor Model

This Example demonstrates that activatable antibodies of the embodiments are able to reduce the growth of MC38 syngeneic tumors.

In this Example, anti-PD1 activatable antibodies J43 MP7-1 2001 m2a EN and J43 MP8-2 2001 m2a EN were analyzed for the ability to reduce the growth of MC38 syngeneic tumors.

The mouse colon carcinoma cell line MC38 was obtained from ATCC. MC38 were grown in RPMI-1640 supplemented with 10% fetal bovine serum at 37° C. in an atmosphere of 5% CO₂ in air. Cells were harvested during the logarithmic growth period, resuspended in PBS with proper cell concentration, and kept on ice for tumor induction.

Each mouse was inoculated subcutaneously at the right flank with 1.5×10⁶ of MC38 cells in PBS for tumor development. The treatments were started when the mean tumor size reached approximately 80 mm³ (60-120 mm³). Tumor sizes were measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm³ using the formula: V=0.5 a×b² where a and b are the long and short diameters of the tumor, respectively.

The mice were grouped and dosed as set forth in Table 22.

TABLE 22 Dose Dose volume Group Count Treatment (mg/kg) (mL/kg) Schedule Route 1 10 mIgG1 MOPC-21 + mIgG12b 10 + 10 10 b.i.w. for IP MPC-11 3 weeks 2 10 Anti-CTLA4 9D9 mIgG2b 10 10 b.i.w. for IP 3 weeks 3 10 Anti-PD-1 (J43 m2a EN) 10 10 b.i.w. for IP 3 weeks 4 10 J43 MP8-2 2001 m2a EN 10 10 b.i.w. for IP 3 weeks 5 10 J43 MP7-1 2001 m2a EN 10 10 b.i.w. for IP 3 weeks 6 10 Anti-PD-1 (J43 m2a EN) + 10 + 10 10 b.i.w. for IP Anti-CTLA4 9D9 mIgG2b 3 weeks 7 10 J43 MP8-2 2001 m2a EN + 10 + 10 10 b.i.w. for IP Anti-CTLA4 9D9 mIgG2b 3 weeks 8 10 J43 MP7-1 2001 m2a EN + 10 + 10 10 b.i.w. for IP Anti-CTLA4 9D9 mIgG2b 3 weeks

FIGS. 31A and 31B, which plots tumor volume versus number of days post initial dose, demonstrates that anti-PD1 activatable antibodies MP7-1 2001 m2a EN and MP8-2 2001 m2a EN inhibit the growth of MC38 syngeneic tumors similar to positive control anti-PD1 antibody J43 m2a EN both as single agents and in combination with anti-CTLA4 antibody 9D9 mIgG2b (BioXCell, West Lebanon, N.H.).

Example 29. Evaluation of Anti-Human PD1 Antibodies and Activatable Anti-Human PD1 Antibodies in Antigen Recall Assay

This Example demonstrates that anti-PD1 antibodies and activated activatable anti-PD1 antibodies of the embodiments (i.e., activatable antibodies in which the CM has been cleaved by a protease) are able to block PD-L1/PD-L2 binding to PD1 and to activate T cells in an antigen recall assay.

As shown in FIGS. 32A-32E, the anti-human PD1 antibody referred to herein as A1.5 Ab (i.e., VH of SEQ ID NO: 21 and VL of SEQ ID NO: 47) blocks PD-L1/PD-L2 binding to PD1 and potently activates T cells in an antigen recall assay. The binding of the anti-human PD-1 A1.5 Ab to immobilized human PD1 as detected by standard plate ELISA is shown in FIG. 32A, and the binding of A1.5 Ab to cynomolgus PD1 (also referred to herein as cyno-PD1, Sino Biological Cat. #90311-CO2H) as detected by ELISA is shown in FIG. 32B. The inhibition of biotinylated human PD-L1 (also referred to herein as biotin-PD-L1) to immobilized PD1 by A1.5 Ab as determined by ELISA is shown in FIG. 32C, and the inhibition of biotinylated human PD-L2 (also referred to herein as biotin-PD-L2) to immobilized PD1 as determined by ELISA is shown in FIG. 32D.

As shown in FIG. 32E, the A1.5 Ab enhances IFN-γ production in a CMV T cell restimulation assay. Briefly, CMV⁺ human PBMCs were plated at 250,000 cells/well with isotype control antibody or with the A1.5 Ab and stimulated with 5 μg/mL CMV lysate for 4 days. Supernatant IFN-γ levels were measured by ELISA.

As shown in FIGS. 33A and 33B, the activatable anti-PD1 antibody referred to herein as A1.5 PD34 2001 (i.e., VH of SEQ ID NO: 21, VL of SEQ ID NO: 47, masking moiety of SEQ ID NO: 99, and cleavable moiety of SEQ ID NO: 214) binds human PD1 with decreased affinity relative to the parental PD-1 Ab, i.e., A1.5 Ab (FIG. 33A), and the A1.5 PD34 2001 activatable antibody shows functional masking in a CMV T cell antigen recall assay (FIG. 33B). Briefly, 4 mg/mL of the A1.5 PD34 2001 activatable antibody was combined with 0 μg/mL or 60 μg/mL recombinant human urokinase (rh uPA, R&D, Cat. #1310-SE) and incubated at 37° C. overnight. Binding of the activatable antibody incubated with uPA to hPD1 was assayed by standard plate ELISA. As shown in FIG. 33, the activatable anti-PD1 antibodies of the disclosure regain full binding activated after activation by a protease.

Example 30. Pharmacokinetic Evaluation of Activatable Anti-Human PD1 Antibodies in Non-Human Primate Model

This Example demonstrates pharmacokinetic and dose proportionality data for anti-PD-1 antibodies and anti-PD-1 activatable antibodies of the embodiments.

Briefly, female cynomolgus monkeys (n=2/group) were dosed with a single IV bolus dose of either the A1.5 antibody or the activatable antibody referred to herein as A1.5 PD34 2001 at 1 mg/kg or at 5 mg/kg. Plasma samples were analyzed for A1.5 and A1.5 PD34 2001 concentrations by a qualified anti-human sandwich ELISA. The results are shown in FIG. 34, where each line represents one individual. Mean PK parameters shown for both A1.5 and A1.5 PD34 2001. Similar results were seen for activatable antibodies A1.5 PD34 2012 and A1.5 PD34 2011 administered to cynomolgus monkeys in a similar study.

Example 31. Activatable Anti-Mouse PD-1 J43 Antibodies Reduce Incidence of Diabetes in NOD Mice Dosed with Anti-CTLA4 Antibody

In this Example, anti-PD-1 J43 activatable antibodies were analyzed for the ability to protect from anti-PD-1 induction of diabetes in NOD mice when dosed concurrently with anti-CTLA-4 9D9 mIgG2b antibody (BioXCell cat #BE0164). The NOD mice, substrain NOD/ShiLtJ, were obtained from Jackson Laboratory at 4 weeks and acclimated on site for 1 week. At 5 weeks, mice were checked for diabetes prior to enrollment, grouped, and dosed as set forth in Table 23. As used herein, the antibody referred to as “J43 m2a EN” (and variations thereof) comprises a heavy chain (HC) of SEQ ID NO: 546; a light chain (LC) of SEQ ID NO: 543; and a constant region of SEQ ID NO: 1516. As used herein, the activatable antibody referred to as “J43 MP8-2 2012 m2a EN” is an activatable antibody comprising the J43 m2a EN antibody, the MP8-2 masking moiety (SEQ ID NO: 549), and the 2012 substrate (SEQ ID NO: 1101), and the activatable antibody referred to as “J43 MP8-2 2001 m2a EN” is an activatable antibody comprising the J43 m2a EN antibody, the MP8-2 masking moiety (SEQ ID NO: 549), and the 2001 substrate (SEQ ID NO: 214).

TABLE 23 Dosing regimen Treatment Dose Dose Article Dose volume Group Count Article #1 (mg/kg) #2 (mg/kg) (mL/kg) Schedule Route 1 8 mIgG1 10 mIgG2b 10 10 d0, d4, d7 IP MOPC-21 MPC-11 2 8 Anti- 10 mIgG1 10 10 d0, d4, d7 IP CTLA4 MOPC- 9D9 21 mIgG2b 3 8 Anti-PD-1 10 mIgG2b 10 10 d0, d4, d7 IP (J43 m2a MPC-11 EN) 4 8 Anti-PD-1 1 mIgG2b 10 10 d0, d4, d7 IP (J43 m2a MPC-11 EN) 5 8 Anti-PD-1 10 Anti- 10 10 d0, d4, d7 IP (J43 m2a CTLA4 EN) 9D9 mIgG2b 6 8 Anti-PD-1 1 Anti- 10 10 d0, d4, d7 IP (J43 m2a CTLA4 EN) 9D9 mIgG2b 7 8 J43 MP8-2 10 mIgG2b 10 10 d0, d4, d7 IP 2012 m2a MPC-11 EN 8 8 J43 MP8-2 10 Anti- 10 10 d0, d4, d7 IP 2012 m2a CTLA4 EN 9D9 mIgG2b 9 8 J43 MP8-2 10 mIgG2b 10 10 d0, d4, d7 IP 2001 m2a MPC-11 EN 10 8 J43 MP8-2 10 Anti- 10 10 d0, d4, d7 IP 2001 m2a CTLA4 EN 9D9 mIgG2b

FIG. 35 which plots % non-diabetic versus number of days post initial dose, shows that the combination of 10 mg/kg anti-PD-1 J43 antibody plus 10 mg/kg anti-CTLA-4 antibody dosed on days 0, 4 and 7 induced diabetes in 50% of NOD mice by day eleven while the same dosing schedule of activatable anti-PD-1 J43 antibodies with CTLA-4 antibody resulted in no induction of diabetes to day eighteen.

Example 32. Activatable Anti-Mouse PD1 J43 m2a EN Antibodies Show Enhanced Efficacy when Coadministered with Anti-CTLA4 Antibody in the MC38 Syngeneic Tumor Model

This Example demonstrates that activatable antibodies of the embodiments are able to reduce the growth of MC38 syngeneic tumors as single agents and induce MC38 tumor regression when coadministered with anti-CTLA4 antibody 9D9 mIgG2b.

In this Example, anti-PD1 activatable antibodies J43 MP8-2 2011 m2a EN and J43 MP8-2 2012 m2a EN were analyzed for the ability to slow the growth of or, in combination with anti-CTLA4 antibody, induce regression in MC38 syngeneic tumors. As used herein, the antibody referred to as “J43 m2a EN” (and variations thereof) comprises a heavy chain (HC) of SEQ ID NO: 546; a light chain (LC) of SEQ ID NO: 543; and a constant region of SEQ ID NO: 1516. As used herein, the activatable antibody referred to as “J43 MP8-2 2011 m2a EN” is an activatable antibody comprising the J43 m2a EN antibody, the MP8-2 masking moiety (SEQ ID NO: 549), and the 2011 substrate (SEQ ID NO: 1100), and the activatable antibody referred to as “J43 MP8-2 2012 m2a EN” is an activatable antibody comprising the J43 m2a EN antibody, the MP8-2 masking moiety (SEQ ID NO: 549), and the 2012 substrate (SEQ ID NO: 1101).

The mouse colon carcinoma cell line MC38 was obtained from ATCC. MC38 cells were grown in RPMI-1640 supplemented with 10% fetal bovine serum at 37° C. in an atmosphere of 5% CO₂ in air. Cells were harvested during the logarithmic growth period, resuspended in PBS with proper cell concentration, and kept on ice for tumor induction.

Each mouse was inoculated subcutaneously at the right flank with 1.5×10⁶ MC38 cells in PBS for tumor development. The treatments were started when the mean tumor size reached approximately 60 mm³ (45-80 mm³). Tumor sizes were measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm³ using the formula: V=0.5 a×b² where a and b are the long and short diameters of the tumor, respectively.

The mice were grouped and dosed as set forth in Table 24.

TABLE 24 Dose Dose volume Group Count Treatment (mg/kg) (mL/kg) Schedule Route 1 10 mIgG1 MOPC-21 + 10 + 10 10 b.i.w. for 3 IP mIgG12b MPC-11 weeks 2 10 Anti-CTLA4 9D9 mIgG2b 10 10 b.i.w. for 3 IP weeks 3 10 Anti-PD-1 (J43 m2a EN) 10 10 b.i.w. for 3 IP weeks 4 10 J43 MP8-2 2012 m2a EN 10 10 b.i.w. for 3 IP weeks 5 10 J43 MP8-2 2011 m2a EN 10 10 b.i.w. for 3 IP weeks 6 10 Anti-PD-1 (J43 m2a EN) + 10 + 10 10 b.i.w. for 3 IP Anti-CTLA4 9D9 mIgG2b weeks 7 10 J43 MP8-2 2012 m2a EN + 10 + 10 10 b.i.w. for 3 IP Anti-CTLA4 9D9 mIgG2b weeks 8 10 J43 MP8-2 2011 m2a EN + 10 + 10 10 b.i.w. for 3 IP Anti-CTLA4 9D9 mIgG2b weeks

FIG. 36, which plots tumor volume versus number of days post initial dose, demonstrates that anti-PD1 activatable antibodies MP8-2 2012 m2a EN and MP8-2 2011 m2a EN inhibit the growth of MC38 syngeneic tumors similar to positive control anti-PD1 antibody J43 m2a EN both as single agents and in combination with anti-CTLA4 antibody 9D9 mIgG2b (BioXCell, West Lebanon, N.H.).

Example 33. Activatable Anti-Mouse PD1 J43 m2a EN Antibodies Show Durable Anti-Tumor Activity when Coadministered with Anti-CTLA4 Antibody in the MC38 Syngeneic Tumor Model

This Example demonstrates anti-PD-1 activatable antibodies of the embodiments in combination with anti-CTLA4 antibodies induce durable anti-tumor complete responses in the MC38 syngeneic tumor model.

In this Example, all animals treated with combinations of anti-CTLA4 and anti-PD1 agents from Example 28 that showed sustained tumor regression ((i) anti-PD-1 J43 m2aEN+anti-CTLA4 9D9 mIgG2b (referred to in FIG. 37 as “anti-PD-1 J43 m2aEN Antibody”+“anti-CTLA4 9D9 mIgG2b Antibody”) (n=8), (ii) activatable antibody anti-PD-1 J43 MP8-2 2012 m2a EN+anti-CTLA4 9D9 mIgG2b (referred to in FIG. 37 as “J43 MP8-2 2012 m2a EN AA”+“anti-CTLA4 9D9 mIgG2b Antibody” where AA stands for activatable antibody) (n=8), and activatable antibody anti-PD-1 J43 MP8-2 2011 m2a EN+anti-CTLA4 9D9 mIgG2b (referred to in FIG. 37 as “J43 MP8-2 2011 m2a EN AA”+“anti-CTLA4 9D9 mIgG2b Antibody” where AA stands for activatable antibody) (n=6)) were implanted at day 38 with 1.5×10⁶ MC38 tumor cells in the left flank opposite to the original MC38 implantation. Five untreated mice were implanted with 1.5×10⁶ MC38 tumor cells to confirm tumorigenic activity of the MC38 cells.

FIG. 37 shows that no mice previously treated with combinations of anti-PD1 and anti-CTLA4 agents showed regrowth of the original tumor sites (right flank) or growth of new tumors (left flank) while all five of the untreated control mice showed rapid tumor growth.

Example 34. Activatable Anti-Mouse PD-1 J43 Antibodies Reduce Incidence of Diabetes in NOD Mice Co-Dosed with Anti-CTLA4 Antibody

This Example demonstrates that anti-PD-1 activatable antibodies of the embodiments in combination with anti-CTLA4 antibodies protected NOD mice from PD1 m2a EN-mediated induction of diabetes.

In this Example, anti-PD-1 J43 activatable antibody MP8-2 2011 m2aEN was analyzed for the ability to protect from anti-PD-1 induction of diabetes in NOD mice when dosed concurrently with anti-CTLA-4 9D9 mIgG2b antibody (BioXcell cat #BE0164). The NOD mice, substrain NOD/ShiLtJ, were obtained from Jackson Laboratory at 4 weeks and acclimated on site for 1 week. At 5 weeks, mice were checked for diabetes prior to enrollment, grouped, and dosed as set forth in Table 25.

TABLE 25 Dosing regimen Treatment Dose Dose Dose volume Group Count Article #1 (mg/kg) Article #2 (mg/kg) (mL/kg) Schedule Route 1 8 Anti-CTLA4 10 mIgG1 10 10 d0, d4, d7 IP 9D9 mIgG2b MOPC-21 2 8 Anti-PD-1 (J43 10 mIgG2b 10 10 d0, d4, d7 IP m2a EN) MPC-11 3 8 Anti-PD-1 (J43 10 Anti-CTLA4 10 10 d0, d4, d7 IP m2a EN) 9D9 mIgG2b 4 8 J43 MP8-2 10 mIgG2b 10 10 d0, d4, d7 IP 2011 m2a EN MPC-11 5 8 J43 MP8-2 10 Anti-CTLA4 10 10 d0, d4, d7 IP 2011 m2a EN 9D9 mIgG2b

FIG. 38, which plots % non-diabetic versus number of days post initial dose, shows that the single agent anti-PD-1 J43 antibody (referred to in the figure as “anti-PD1 J43 Ab”) and the combination of 10 mg/kg anti-PD-1 J43 antibody plus 10 mg/kg anti-CTLA4 antibody (referred to in the figure as “anti-PD1 J43 Ab”+“anti-CTLA4 9D9 mIgG2b”) dosed on days 0, 4 and 7 induced diabetes in 25% of NOD mice by day nine. In contrast, the same dosing schedule of activatable antibody anti-PD-1 J43 MP8-2 2011 m2a EN (referred to in the figure as “J43 MP8-2 2011 m2a EN AA” where AA stands for activatable antibody) as a single agent or in combination with anti-CTLA4 antibody (referred to in the figure as “J43 MP8-2 2011 m2a EN AA+“anti-CTLA4 9D9 mIgG2b” where AA stands for activatable antibody) resulted in no induction of diabetes to day fifteen.

Example 35. Anti-PD-1 Nivolumab Activatable Antibodies of the Embodiments are Functionally Masked in a Human T-Cell Restimulation Assay

This Example describes the effect of masking moieties on the biological function of the anti-PD-1 nivolumab antibody.

PBMCs from a CMV-positive donor (Hemacare) were plated at 2×10⁵ cells/well in the presence of CMV viral lysate (Astarte) and either anti-PD-1 antibody A1.5, anti-PD-1 activatable antibody A1.5 PD34 2001, anti-PD-1 antibody nivolumab (NV1), anti-PD-1 nivolumab activatable antibody NV1 NV07 2001, or an hIgG4 isotype control antibody. After four days, supernatant was removed from each well and IFN-gamma levels were assayed using an IFN-gamma ELISA kit (Life Technologies, Carlsbad, Calif.).

FIG. 39 demonstrates that the anti-PD-1 activatable antibody A1.5 PD34 2001 (referred to in the figure as “A1.5 PD34 2001 AA” where AA stands for activatable antibody) and anti-PD1 nivolumab activatable antibody NV1 NV07 2001 (referred to in the figure as “NV1 NV07 2001 AA” where AA stands for activatable antibody) effected increased CMV-stimulated IFN-gamma secretion compared to the control hIgG4 antibody (referred to in the figure as hIgG4) but decreased potency relative to anti-PD-1 parental antibody A1.5 (referred to in the figure as A1.5) or anti-PD-1 parental antibody nivolumab (referred to in the figure as NV1).

Example 36. Anti-PD-1 Pembrolizumab Activatable Antibodies of the Embodiments are Functionally Masked in a Human T-Cell Restimulation Assay

This example describes the effect of masking moieties on the biological function of the anti-PD-1 pembrolizumab antibody.

PBMCs from a CMV-positive donor (Hemacare) were plated at 2×10⁵ cells/well in the presence of 4 ug/ml CMV viral lysate (Astarte) and either anti-PD-1 antibody A1.5, anti-PD-1 antibody pembrolizumab (PM1), anti-PD-1 pembrolizumab activatable antibody PM1 PM07 2001, or an hIgG4 isotype control antibody. After four days, supernatant was removed from each well and IFN-gamma levels were assayed using an IFN-gamma ELISA kit (Life Technologies, Carlsbad, Calif.).

FIG. 40 demonstrates that anti-PD1 pembrolizumab activatable antibody PM1 PM07 2001 (referred to in the figure as “PM1 PM07 2001 AA” where AA stands for activatable antibody) effected increased CMV-stimulated IFN-gamma secretion compared to the control hIgG4 antibody (referred to in the figure as hIgG4) but decreased potency relative to anti-PD-1 parental antibody A1.5 (referred to in the figure as A1.5) or anti-PD-1 parental antibody pembrolizumab (referred to in the figure as PM1).

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following. 

1-99. (canceled)
 100. An isolated antibody or antigen binding fragment thereof (AB) that specifically binds to a human PD-1, wherein the AB comprises: a VH CDR1 amino acid sequence comprising GFTFSNYAMS SEQ ID NO: 655), a VH CDR2 amino acid sequence comprising YISNGGGDTH (SEQ ID NO: 660), a VH CDR3 amino acid sequence comprising ENYGTSPFVY (SEQ ID NO: 666), a VL CDR1 amino acid sequence comprising RASESVDNYGIS (SEQ ID NO: 673), a VL CDR2 amino acid sequence comprising AASNQGS (SEQ ID NO: 678), and a VL CDR3 amino acid sequence comprising QQSKDVPWT (SEQ ID NO: 683).
 101. An activatable antibody that, when activated, specifically binds to a human PD-1, wherein said activatable antibody comprises: an antibody or an antigen binding fragment thereof (AB) that specifically binds to mammalian PD-1, wherein the AB comprises a VH CDR1 amino acid sequence comprising GFTFSNYAMS SEQ ID NO: 655), a VH CDR2 amino acid sequence comprising YISNGGGDTH (SEQ ID NO: 660), a VH CDR3 amino acid sequence comprising ENYGTSPFVY (SEQ ID NO: 666), a VL CDR1 amino acid sequence comprising RASESVDNYGIS (SEQ ID NO: 673), a VL CDR2 amino acid sequence comprising AASNQGS (SEQ ID NO: 678), and a VL CDR3 amino acid sequence comprising QQSKDVPWT (SEQ ID NO: 683); a masking moiety (MM) that inhibits the binding of the AB to mammalian PD-1 when the activatable antibody is in an uncleaved state; and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease, wherein the MM is coupled to the AB via the CM.
 102. The activatable antibody of claim 101, wherein the AB specifically blocks a human PD-L1 and a human PD-L2 from binding to the human PD-1.
 103. The activatable antibody of claim 101, wherein the activatable antibody in an uncleaved state specifically binds to the human PD-1 with a dissociation constant of 0.5 nM to 1 nM.
 104. The activatable antibody of claim 101, wherein the CM is a substrate for a protease that is active in diseased tissue.
 105. A pharmaceutical composition comprising the activatable antibody of claim 101 and a carrier.
 106. An isolated nucleic acid molecule encoding the activatable antibody of claim
 101. 107. A vector comprising the isolated nucleic acid molecule of claim
 106. 108. A method of producing an activatable antibody by culturing a cell under conditions that lead to expression of the activatable antibody, wherein the cell comprises the nucleic acid molecule of claim
 106. 109. A method of reducing binding of a ligand selected from the group consisting of PD-L1 or PD-L2 to PD-1 on T cells comprising administering an effective amount of the activatable antibody of claim 101 to a subject in need thereof.
 110. A method of reducing immune suppression mediated by engagement of PD-1 on T cells to PD-L1 or PD-L2 on tumor or other immune cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of the activatable antibody of claim
 101. 111. A method of treating, alleviating a symptom of, or delaying the progression of a cancer comprising administering to a subject in need thereof a therapeutically effective amount of the activatable antibody of claim
 101. 112. The activatable antibody of claim 101, wherein the activatable antibody comprises a heavy chain that comprises an IgG4 amino acid sequence.
 113. The activatable antibody of claim 101, wherein the AB comprises a VH comprising the amino acid sequence of SEQ ID NO:
 33. 114. The activatable antibody of claim 101, wherein the AB comprises a VL comprising the amino acid sequence of SEQ ID NO:
 41. 115. The activatable antibody of claim 101, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-213, 384-514, and 548-571.
 116. The activatable antibody of claim 101, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 294-361, 1092-1112, 1157, 1158, 1161, 1162, 1165, 1166, 1169, 1520, and 1695-1704.
 117. The activatable antibody of claim 101, wherein the AB has one or more of the characteristics selected from the group consisting of: (a) the AB is an antigen binding fragment selected from the group consisting of a Fab fragment, a F(ab′)2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody; (b) the AB is a monoclonal antibody; (c) the AB is linked to the CM; (d) the AB is linked directly to the CM; and (e) the AB is linked to the CM via a linking peptide.
 118. A conjugated activatable antibody comprising the activatable antibody of claim 101 conjugated to an agent.
 119. The conjugated activatable antibody of claim 118, wherein the agent has one or more of the following characteristics selected from the group consisting of: (a) the agent is conjugated to the activatable antibody via a linker; (b) the agent is conjugated to the activatable antibody via a cleavable linker, (c) the linker is conjugated to the activatable antibody via a non-cleavable linker; (d) the agent is a detectable moiety; and (e) the agent is a diagnostic agent. 